Spectra Plus 5.0 Keygen
A complex signal in the time domain looks vastly different than in the frequency domain. The time-domain measurement shows an impure sine wave. Without measuring in the frequency domain, the source and frequency of the second harmonic remain unknown. Spectrum analysis uncovers sources of interference by displaying the spectral components independently. The time-domain still provides useful information, such as the pulse rise and fall times of a signal, but the frequency domain allows you to determine the harmonic content of a signal, such as out-of-band emissions and distortion. For more information refer to this blog: Spectrum Analysis Basics, Part 1 - What is a Spectrum Analyzer?
Spectra Plus 5.0 Keygen
A xenograft colorectal carcinoma mouse model was established by injecting 1107 VCR-sensitive or resistant HCT-8 cells subcutaneously in the flank region of each female nude BALB/c mice (4-week-old, Vital River Lab). When tumors reached about 100 mm3, the nude mice were randomized into four groups (n = 6) according to tumor volumes and body weights. Drugs were injected intraperitoneally daily for 27 days, including IVM (2 mg/kg/day), VCR (0.2 mg/kg/day), IVM (2 mg/kg/day) plus VCR (0.2 mg/kg/day). To prepare IVM for injection, a stock solution (5 mg/ml in DMSO) was prepared and then diluted by using 0.9% NaCl, which resulted in a homogeneous suspension of IVM. Two hundred microliters (200 μl) of the IVM was injected to each mouse. VCR was also prepared in 0.9% NaCl, and mice injected with only 0.9% NaCl solution served as vehicle control. Tumor volume was measured every three days by using calipers. Tumor volumes were calculated as V = length width2/2. On the 27th day, the tumors were harvested, weighed, and then fixed in 4% paraformaldehyde for immunofluorescence and immunohistochemistry analysis.
The cells were lysed and then centrifuged. The supernatants were incubated with the anti-avermectins (AVMs) antibody, which had a cross-reactivity of 100% with abamectin (ABM) and 25% with IVM  (provided by Dr. Jianzhong Shen) at 4C in rotation overnight. Then 80 μl of protein G plus A agarose (Beyotime Biotechnology, Jiangsu, China) was added and the mixture was incubated at 4C in rotation for another 6 h. Then, the immunocomplexes were washed and the precipitated beads were resuspended in 50 μl of 2 loading buffer for the electrophoresis.
To evaluate whether IVM can suppress tumorigenesis in vivo, we used a human tumor xenograft model by subcutaneously injecting VCR-sensitive (S) or resistant (R) HCT-8 cells into the dorsal flank of female nude mice. The inhibitory effect of VCR on the growth of the tumors derived from R cells (simplified as R tumors) was weaker than that of the tumors derived from S cells (simplified as S tumors) (Fig. 2a and b). And after treated with VCR plus IVM, the growth rate of the S and R tumors was reduced compared with those with VCR alone treatment (Fig. 2a and b). In addition, the tumor weight (Fig. 2c) and the tumor size (Fig. 2d) from the mice treated with VCR plus IVM was much lower or smaller than those of the mice treated with VCR alone. These results indicated that IVM not only significantly reversed the resistance of R tumors to VCR but also strongly ameliorated the response of S tumors to VCR in vivo.
To determine whether IVM can suppress tumorigenesis of non-solid tumor e.g. leukemia in vivo, we established a human tumor xenograft model by injecting ADR-sensitive (SK)/resistant (RK) K562 cells into the peripheral blood of male NOD/SCID mice via tail vein. The survival curves indicated that the survival percentage of the SK mice treated with ADR plus IVM was higher than that of the mice treated with vehicle. Although there was no statistical significance between RK mice treated with ADR plus IVM and those treated with vehicle, the overall survival percentage was higher in ADR plus IVM treatment group in RK mice (Fig. 3a). The body weight of the mice in ADR plus IVM treatment groups had almost no significant change throughout the experiment, while the body weight severely declined in the vehicle group (Additional file 1: Figure S2A), and the relative weight of spleen was almost restored to the normal level by IVM plus ADR treatments (Additional file 1: Figure S2B). These findings indicated that the RK leukemia was indeed resistant to ADR treatment, and co-treatment with IVM significantly enhanced the anticancer activity of ADR to both SK and RK leukemia.
In addition, we found that ADR alone treatment significantly decreased the K562 cell numbers in peripheral blood and spleen only in the mice with SK leukemia, but not RK leukemia (Fig. 3b-e). However, IVM plus ADR treatment decreased the number of K562 cells in both peripheral blood and spleen compared with the vehicle or ADR alone treatment in not only the mice with SK leukemia but also those with RK leukemia (Fig. 3b-e). Moreover, the cells with positive staining of CD13 or CD33, the surface markers of K562 cells, as well as the mRNA levels of bcr/abl fusion gene, a marker of chronic myeloid leukemia, in peripheral blood and bone marrow of IVM plus ADR-treated mice were lower than that in the ADR alone-treated mice (Fig. 3f; Additional file 1: Figure S2C & D). The above results indicated that IVM enhanced the anti-tumor effect of ADR in leukemia, and drastically reversed the resistance of leukemia to ADR in vivo.
The IC50 value of VCR in the R cells with P-gp knocked-down (150.01 nM) was significantly lower than that in the R cells without P-gp knocked-down (1015.52 nM), which indicated that P-gp was essential for the multidrug resistance in the R cells (Additional file 1: Figure S3A & B; Fig. 1a). Thus, we then determined whether IVM altered P-gp expression in HCT-8 cells. The mRNA and protein levels of MDR1/P-gp in the R cells were indeed higher than that in the S cells, and mRNA and protein levels of MDR1/P-gp were decreased by IVM in both S and R cells (Fig. 4a and b). In addition, after treatment of VCR plus IVM, the intracellular level of VCR increased compared with that in the cells treated with VCR alone (Fig. 4c). Altogether, these results suggested that IVM inhibited the expression and function of P-gp.
Furthermore, the inhibited cell viability of both S and R cells by the chemicals treatment was further reduced in the P-gp knocked-down cells (Fig. 4d). The IVM-reduced P-gp expression was recovered by the overexpression of P-gp or the treatment of sulforaphane (SFP), an activator of the transcription factor Nrf2, which could induce the expression of P-gp  (Additional file 1: Figure S3C & D), and the viability of the cells treated with IVM plus VCR increased compared with that of the cells without P-gp overexpression or SFP treatment, and this effect was more obvious in the R cells than that in the S cells (Fig. 4e; Additional file 1: Figure S3E). Thus, these results indicated that P-gp overexpression played a very important role in VCR resistance, and IVM could increase the sensitivity of the cells to VCR by inhibiting P-gp expression.
When the cells were treated with VCR plus IVM in the presence of EGF or with EGFR overexpression, the cell viability increased compared with the cells treated with VCR plus IVM in the absence of EGF or without EGFR overexpression (Fig. 5f and g), whereas the treatment of EGFR inhibitor LAP or knockdown of EGFR further decreased the cell viability, which was inhibited by VCR plus IVM treatment (Fig. 5h and i). Thus, IVM could increase the sensitivity of the cells to VCR by inhibiting EGFR. In order to prove that the reversal effects of IVM was indeed mediated by the inhibition of EGFR, we treated the HCT-116 cells and EGFR knockout HCT-116 cells with VCR and IVM. We found that the IC50 value of IVM and VCR was not significantly different between the wild type cells and the EGFR knockout cells (Fig. 6a). IVM increased the sensitivity of the HCT-116 cells to VCR in a dose-dependent manner, which was consistent with the results in HCT-8 cells; however, IVM could not increase the sensitivity of the EGFR knockout cells to VCR (Fig. 6b). As shown in Fig. 6c and d, the expression levels of the proteins p-EGFR and P-gp and the mRNA level of MDR1 were decreased by IVM alone or in combination with VCR in the wild type cells; while those were not altered by the treatment in the EGFR knockout cells. The similar result was observed in the cells with LAP treatment (Fig. 6e-g). Thus, the effect of IVM on P-gp expression was mainly mediated by EGFR. In addition, when HCT-8 cells were pretreated with IVM and then treated with VCR in presence or absence of IVM, the cells treated with VCR alone had lower cell viability compared with those cells treated with only VCR but without IVM pretreatment, which indicated that the reversal effect of IVM on the resistance of the cells to the drug still existed even after IVM was removed from the medium; however, pretreatment with VRP, a classical inhibitor of P-gp, did not change the viability of the cells treated with VCR alone, indicating that the reversal effect of VRP was present only when VRP was used at the same time with VCR (Fig. 6h). This result supported the notion that the reversal effect of IVM was not mediated by its direct inhibition of P-gp activity as the classical P-gp inhibitor VRP did. Altogether, the reversal effect of IVM on the resistance of the cells to the drug was largely mediated by the inhibition of EGFR phosphorylation, not by the direct inhibition of P-gp.
We then sought to determine the downstream molecules of the inhibition of EGFR by IVM. We found that IVM inhibited ERK and Akt phosphorylation (Fig. 7a). Treatment with EGF or overexpression of EGFR stimulated the phosphorylation of ERK and Akt, which indicated that ERK and Akt were downstream of EGFR (Additional file 1: Figure S4A & B). Akt or ERK were constitutively activated by using adenoviral vectors Ad-Akt-myr and Ad-MKK1-R4F (MKK1 is the upstream kinase for ERK and could phosphorylate and stimulate ERK in the cells), respectively. Then, after the activation of Akt or ERK, the decrease of P-gp expression triggered by IVM was reduced (Fig. 7b and c), and the viability of the cells treated with VCR plus IVM was induced (Fig. 7d and e). In addition, treatment with a PI3K/Akt inhibitor wortmannin or an ERK inhibitor U0126 further inhibited the IVM-repressed P-gp expression and cell viability (Additional file 1: Figure S4C-F). Furthermore, we found that the ERK inhibitor U0126 also inhibited the activation of Akt, but the Akt inhibitor wortmannin did not inhibit ERK activation (Additional file 1: Figure S4G & H). Thus, ERK acted upstream of Akt. Indeed, activation of ERK by Ad-MKK1 also activated Akt, whereas activation of Akt by Ad-Akt did not activate ERK (Fig. 7b and c). In sum, these findings suggested that the increased sensitivity of cells to chemotherapeutic drugs by IVM was mediated by inhibiting the expression of P-gp via inhibiting the EGFR/ERK/Akt pathway.