By expressing two genes (hTERT and Cdk4), we have developed a method to reproducibly generate continuously replicating human bronchial epithelial cell (HBEC) lines that provide a novel resource to ...study the molecular pathogenesis of lung cancer and the differentiation of bronchial epithelial cells. Twelve human bronchial epithelial biopsy specimens obtained from persons with and without lung cancer were placed into short-term culture and serially transfected with retroviral constructs containing cyclin-dependent kinase (Cdk) 4 and human telomerase reverse transcriptase (hTERT), resulting in continuously growing cultures. The order of introduction of Cdk4 and hTERT did not appear to be important; however, transfection of either gene alone did not result in immortalization. Although they could be cloned, the immortalized bronchial cells did not form colonies in soft agar or tumors in nude mice. The immortalized HBECs have epithelial morphology; express epithelial markers cytokeratins 7, 14, 17, and 19, the stem cell marker p63, and high levels of p16(INK4a); and have an intact p53 checkpoint pathway. Cytogenetic analysis and array comparative genomic hybridization profiling show immortalized HBECs to have duplication of parts of chromosomes 5 and 20. Microarray gene expression profiling demonstrates that the Cdk4/hTERT-immortalized bronchial cell lines clustered together and with nonimmortalized bronchial cells, distinct from lung cancer cell lines. We also immortalized several parental cultures with viral oncoproteins human papilloma virus type 16 E6/E7 with and without hTERT, and these cells exhibited loss of the p53 checkpoint and significantly different gene expression profiles compared with Cdk4/hTERT-immortalized HBECs. These HBEC lines are a valuable new tool for studying of the pathogenesis of lung cancer.
Retroviral vectors including lentiviral vectors are commonly used tools to stably express transgenes or RNA molecules in mammalian cells. Their utilities are roughly divided into two categories, ...stable overexpression of transgenes and RNA molecules, which requires maximal transduction efficiency, or functional selection with retrovirus (RV)-based libraries, which takes advantage of retroviral superinfection resistance. However, the dynamic features of RV-mediated transduction are not well characterized. Here, we engineered two murine stem cell virus-based retroviral vectors expressing dual fluorescence proteins and antibiotic markers, and analyzed virion production efficiency and virion stability, dynamic infectivity and superinfection resistance in different cell types, and strategies to improve transduction efficiency. We found that the highest virion production occurred between 60 and 72 h after transfection. The stability of the collected virion supernatant decreased by >60% after 3 days in storage. We found that RV infectivity varied drastically in the tested human cancer lines, while low transduction efficiency was partially overcome with increased virus titer, prolonged infection duration and/or repeated infections. Furthermore, we demonstrated that RV receptors PIT1 and PIT2 were lowly expressed in the analyzed cells, and that PIT1 and/or PIT2 overexpression significantly improved transduction efficiency in certain cell lines. Thus, our findings provide resourceful information for the optimal conditions of retroviral-mediated gene delivery.
Human gastric carcinoma shows a higher prevalence of microsatellite instability (MSI) than does any other type of sporadic human cancer. The reasons for this high frequency of MSI are not yet known. ...In contrast to endometrial and colorectal carcinoma, mutations of the DNA mismatch repair (MMR) genes hMLH1 or hMSH2 have not been described in gastric carcinoma. However, hypermethylation of the hMLH1 MMR gene promoter is quite common in MSI-positive endometrial and colorectal cancers. This hypermethylation has been associated with hMLH1 transcriptional blockade, which is reversible with demethylation, suggesting that an epigenetic mechanism underlies hMLH1 gene inactivation and MMR deficiency. Therefore, we studied the prevalence of hMLH1 promoter hypermethylation in a total of 65 gastric tumors: 18 with frequent MSI (MSI-H), 8 with infrequent MSI (MSI-L), and 39 that were MSI negative. We found a striking association between hMLH1 promoter hypermethylation and MSI; of 18 MSI-H tumors, 14 (77.8%) showed hypermethylation, whereas 6 of 8 MSI-L tumors (75%) were hypermethylated at hMLH1. In contrast, only 1 of 39 (2.6%) MSI-negative tumors demonstrated hMLH1 hypermethylation (P<0.0001 for MSI-H or MSI-L versus MSI-negative). Moreover, hypermethylated cancers demonstrated diminished expression of hMLH1 protein by both immunohistochemistry and Western blotting, whereas nonhypermethylated tumors expressed abundant hMLH1 protein. These data indicate that hypermethylation of hMLH1 is strongly associated with MSI in gastric cancers and suggest an epigenetic mechanism by which defective MMR occurs in this group of cancers.
Background
Patients with posttraumatic stress disorder (PTSD) often share co‐morbidity with chronic pain conditions. Recent studies suggest a role of P2X3 receptors and ATP signaling in pain ...conditions. However, the underlying mechanisms of visceral hyperalgesia following exposure to PTSD‐like stress conditions remain unclarified.
Methods
The behavior and hormones relevant for PTSD were studied. Visceromotor responses (VMR) and the abdominal withdrawal reflexes (AWR) to colorectal distention (CRD) were recorded to determine P2X3‐receptor–mediated alteration of hyperalgesia following single‐prolonged stress (SPS) exposure. Immunofluorescence, Western blotting, and patch‐clamp were used.
Key Results
The escape latency, adrenocorticotropic hormone and cortisol were increased on days 7–14. Visceromotor responses and AWR was reduced at day 1 in SPS rats but increased to higher levels than in controls after exposure to day 7. Intrathecal administration of the P2X3‐receptor antagonist TNP‐ATP abolished the CRD response. Based on immunofluorescence and Western blotting analysis, SPS‐treated rats exhibited reduced P2X3 expression in dorsal root ganglia (DRG) after day 1 compared with controls. P2X3 expression in DRG was enhanced on day 7 after SPS and the increase of the P2X3 expression was maintained on day 14 and 21 compared with controls. The P2X3‐receptor agonist α,β‐me ATP (10 μM) induced a fast desensitizing inward current in DRG neurons of both control and SPS‐treated rats. The average peak current densities in SPS‐treated group were increased 3.6‐fold. TNP‐ATP (100 nM) markedly blocked all fast α,β‐me ATP‐induced inward currents in the DRG neurons both in control and SPS‐treated rats.
Conclusions & Inferences
The data indicate an important role of P2X3 signaling in visceral hyperalgesia following PTSD‐like stress.
Patients with posttraumatic stress disorder share co‐morbidity with chronic pain conditions. The purpose was to study the underlying mechanisms of increased pain sensitivity in rats following exposure to posttraumatic stress disorder conditions. Motoric responses to colorectal distention were reduced initially in stress‐exposed rats but increased afterwards to higher levels. Stress‐exposure initially exhibited reduced dorsal root ganglion P2X3 ionic channel expression, which was followed by increased P2X3 expression. The data indicate an important role of P2X3 signaling in visceral pain.
The matrix effect of powder samples, especially for soil samples, is significant in laser-induced breakdown spectroscopy (LIBS), which affects the prediction accuracy of the element concentration. In ...order to reduce this effect of the soil samples in LIBS, the standard addition method (SAM) based on background removal by wavelet transform algorithm was investigated in this work. Five different kinds of certified reference soil samples (lead (Pb) concentrations were 110, 283, 552, 675, and 1141 ppm, respectively) were used to examine the accuracy of this method. The root mean square error of prediction (RMSEP) was more than 303 ppm by using the conventional calibration method. After adoption of SAM with background removal by wavelet transform algorithm, the RMSEP was reduced to 25.7 ppm. Therefore, the accuracy of the Pb element was improved significantly. The mechanism of background removal by wavelet transform algorithm based on SAM is discussed. Further study demonstrated that this method can also improve the predicted accuracy of the Cd element.
Alteration in cellular prion protein (PrP
C
) localization on the cell surface through mediation of the glycosylphosphatidylinositol (GPI) anchor has been reported to dramatically affect the ...formation and infectivity of its pathological isoform (PrP
Sc
). A patient with Gerstmann-Sträussler-Scheinker (GSS) syndrome was previously found to have a nonsense heterozygous PrP-Q227X mutation resulting in an anchorless PrP. However, the allelic origin of this anchorless PrP
Sc
and cellular trafficking of PrP
Q227X
remain to be determined. Here, we show that PrP
Sc
in the brain of this GSS patient is mainly composed of the mutant but not wild-type PrP (PrP
Wt
), suggesting pathological PrP
Q227X
is incapable of recruiting PrP
Wt
in vivo. This mutant anchorless protein, however, is able to recruit PrP
Wt
from humanized transgenic mouse brain but not from autopsied human brain homogenates to produce a protease-resistant PrP
Sc
-like form in vitro by protein misfolding cyclic amplification (PMCA). To further investigate the characteristics of this mutation, constructs expressing human PrP
Q227X
or PrP
Wt
were transfected into neuroblastoma cells (M17). Fractionation of the M17 cells demonstrated that most PrP
Wt
is recovered in the cell lysate fraction, while most of the mutant PrP
Q227X
is recovered in the medium fraction, consistent with the results obtained by immunofluorescence microscopy. Two-dimensional gel-electrophoresis and Western blotting showed that cellular PrP
Q227X
spots clustered at molecular weights of 22–25 kDa with an isoelectric point (
p
I) of 3.5–5.5, whereas protein spots from the medium are at 18–26 kDa with a
p
I of 7–10. Our findings suggest that the role of GPI anchor in prion propagation between the anchorless mutant PrP and wild-type PrP relies on the cellular distribution of the protein.
Ciprofol is a newly developed intravenous sedative-hypnotic drug. The objective of the study was to prove whether ciprofol was non-inferior to propofol for the successful induction of general ...anesthesia. The ideal post-induction sedation level was assessed by comparing patients' clinical symptoms and their hemodynamic effects in responding to noxious stimuli, mostly tracheal intubation and bispectral index (BIS) alterations following ciprofol/propofol administration.
In this multi-center, randomized, double-blind phase 3 trial, selective surgery patients were randomly assigned in a 1:1 ratio to either ciprofol 0.4 mg/kg (n = 88) or propofol 2.0 mg/kg (n = 88) groups. The primary endpoint was the percentage of patients with successful anesthesia inductions. Secondary endpoints included the times to successful induction of general anesthesia and loss of the eyelash reflex, changes in BIS, as well as safety indicators.
The anesthesia induction success rates for both ciprofol 0.4 mg/kg and propofol 2 mg/kg groups were 100.0%, with a 95% CI lower success limit of -4.18% difference between the two groups, indicating that ciprofol was non-inferior to propofol. For secondary outcomes, the average time to successful anesthesia and loss of the eyelash reflex were 0.91 min and 0.80 min for ciprofol and 0.80 min and 0.71 min for propofol, respectively. The pattern of BIS changes with ciprofol was similar to propofol and stable during the anesthesia maintenance period. Safety was comparable with 88.6% TEAEs in the ciprofol group compared to 95.5% in the propofol group. The incidence of injection pain was significantly lower in the ciprofol group compared to the propofol group (6.8% vs. 20.5%, p < 0.05). In addition, the patients treated with ciprofol had a lesser increase in blood pressure and heart rate, and fewer cases with BIS > 60 within 15 min of intravenous administration, which indicated that ciprofol may provide a better ideal sedation level during the post-induction period under an equivalent dosing regimen to propofol.
Ciprofol for patients undergoing selective surgery is a new option for the induction of general anesthesia.
Ferroelectrics exhibit polarization tunable resistance switching behaviors, which are promising for next-generation non-volatile memory devices. For technological applications, thinner nanoscale ...arrays are expected, which feature with higher density and larger ON/OFF (RON/OFF) ratios in the metal/ferroelectrics/semiconductor heterojunction. Here, we acquire high density BiFeO3 (BFO) nano-islands around 10 nm in thickness displaying a high RON/OFF ratio of 103, comparable to the tunnel junctions. Moreover, both the macroscopic and microscopic resistive switching behaviors of the present BFO films reveal an unexpected filamentary-type resistive switching which is modified by the charged domain walls in nano-islands dominated by the center-type domains. Particularly, the charged domain walls spontaneously formed within the BFO nano-islands are proposed as the conductive paths based on the redistribution of carriers under the applied voltages. Potential applications for memories with large RON/OFF ratios of such kind of configurable charged domain walls are demonstrated.
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