Comparisons of ELISA and Western blot assays for detection of autophagy flux

We analyzed autophagy/mitophagy flux in vitro (C2C12 myotubes) and in vivo (mouse skeletal muscle) following the treatments of autophagy inducers (starvation, rapamycin) and a mitophagy inducer (carbonyl cyanide m-chlorophenylhydrazone, CCCP) using two immunodetection methods, ELISA and Western blotting, and compared their working range, accuracy, and reliability. The ELISAs showed a broader working range than that of the LC3 Western blots (Table 1). Table 2 showed that data value distribution was tighter and the average standard error from the ELISA was much smaller than those of the Western blot, directly relating to the accuracy of the assay. Test-retest reliability analysis showed good reliability for three individual ELISAs (interclass correlation, ≥ 0.7), but poor reliability for three individual Western blots (interclass correlation, ≤ 0.4) (Table 3). Keywords: Autophagy, Mitophagy, ELISA, Western blot, Skeletal muscl

Bone regeneration potential of sub-microfibrous membranes with osteogenic induction of rBMSC for tissue engineering

Purpose: To examine the biocompatibility and osteoinductive potential of  sub-microfibrous membranes with cells in vitro and in vivo.Methods: Polylactic acid (PLA) and poly-ε-caprolactone (PCL) were blended at various volume ratios (PLA:PCL = 100:0, 70:30, 50:50, 30:70 and 0:100) and each membrane form was prepared by electrospinning. Cell viability,  biocompatibility, and bone regeneration were measured.Results: The membranes from the PLA/PCL blends prepared by an electrospinning process showed a range of diameter distribution ranging from 1,580 to 550 nm. The cells of 100 % PCL membrane (smallest diameter) exhibited significantly higher adhesion and proliferation than those of the other membranes. Among the  membranes from PLA/PCL blends, PCL membrane showed weak inflammatory changes in the early stages of implantation without acute or chronic inflammation. PCL membranes with osteogenically-induced cells successfully stimulated new bone formation in a rate calvarial defect model.Conclusion: The results indicate that biodegradable PCL sub-microfibrous membrane produced by electrospinning process seems to have excellent biocompatibility, and may be used as a scaffold for bone tissue engineering.Keywords: Biocompatibility, Hard tissue, Biomaterial availability, Bone remodeling, Polylactic acid, Poly-ε-caprolactone, Osteoinductive potential, Sub-microfibrous membrane

Inhibition of ATR Increases the Sensitivity to WEE1 Inhibitor in Biliary Tract Cancer

Purpose Currently, the DNA damage response (DDR) pathway represents a key target for new cancer drug development. Advanced biliary tract cancer (BTC) has a poor prognosis because of the lack of efficacious treatment options. Although DNA repair pathway alterations have been reported in many patients with BTC, little is known regarding the effects of DDR-targeted agents against BTC. Materials and Methods In this study, nine BTC cell lines were exposed to the WEE1 inhibitor (AZD1775). In vitro, MTT assay, colony-forming assay, cell cycle analysis, phospho-histone H3 staining assay, Transwell migration assay, and western blot were performed. Then, to enhance the antitumor effect of AZD1775, the combination treatment of WEE1 inhibitor and ataxia telangiectasia mutated and Rad3 related (ATR) inhibitor (AZD6738) was conducted using MTT assay and comet assay. Finally, HuCCT-1 and SNU2670 xenograft models were established to confirm the anti-tumor effect of AZD1775 alone. Furthermore, the combination treatment was also evaluated in SNU2670 xenograft models. Results AZD1775 blocked the phosphorylation of CDC2 and CDC25C in all cell lines, but significantly increased apoptosis and S phase arrest in sensitive cells. However, increased p-ATR and phosphorylated ataxia telangiectasia mutated levels were observed in less sensitive cells. In addition, in vitro and in vivo data illustrated that AZD1775 combined with AZD6738 exerted more potent anti-tumor effects than either drug alone. Although WEE1 inhibition has promising anti-tumor effects in some BTC cells, the addition of ATR inhibitors could enhance its efficacy. Conclusion Taken together, this study supports further clinical development of DDR-targeted strategies as monotherapy or combination regimens for BTC.

Effects on Growth and Osteogenic Differentiation of Mesenchymal Stem Cells by the Zinc-Added Sol-Gel Bioactive Glass Granules

Responses of mesenchymal stem cells (MSCs) cultured with zinc-added (2 and 5%) bioactive glass granules were evaluated in terms of cell growth and osteogenic differentiation. MSCs were cultured with different quantities (3, 10 and 30) of glass granules for up to 21 days in the osteogenic medium. Cell growth was stimulated by a small quantity of glasses, particularly those that contained zinc. Osteogenic differentiation, as assessed by alkaline phosphatase activity (ALP) activity, was significantly enhanced by the glasses, particularly with large quantities of glass and for prolonged culturing. Expression of bone-sialo protein (BSP) was significantly up-regulated around the bioactive glass granules. Moreover, the zinc addition significantly altered the ALP and BSP depending on the culture time and glass quantity. Cellular mineralization was improved in all glass samples, and particularly in the 2% zinc-glass. Taken together, the zinc addition to bioactive glass induced the MSCs growth and their osteogenic differentiation, at least to the level of zinc-free glass, and with even higher level observed depending on the quantity and culture time. These findings indicate that the zinc addition to bioactive glass may be useful in development of biomaterials for the stimulation of adult stem cell in bone tissue engineering

Therapeutic Co-targeting of WEE1 and ATM Downregulates PD-L1 Expression in Pancreatic Cancer

Purpose Pancreatic cancer (PC) is one of the most lethal cancers worldwide, but there are currently no effective treatments. The DNA damage response (DDR) is under investigation for the development of novel anti-cancer drugs. Since DNA repair pathway alterations have been found frequently in PC, the purpose of this study was to test the DDR-targeting strategy in PC using WEE1 and ATM inhibitors. Materials and Methods We performed in vitro experiments using a total of ten human PC cell lines to evaluate antitumor effect of AZD1775 (WEE1 inhibitor) alone or combination with AZD0156 (ATM inhibitor). We established Capan-1-mouse model for in vivo experiments to confirm our findings. Results In our research, we found that WEE1 inhibitor (AZD1775) as single agent showed anti-tumor effects in PC cells, however, targeting WEE1 upregulated p-ATM level. Here, we observed that co-targeting of WEE1 and ATM acted synergistically to reduce cell proliferation and migration, and to induce DNA damage in vitro. Notably, inhibition of WEE1 or WEE1/ATM downregulated programmed cell death ligand 1 expression by blocking glycogen synthase kinase-3 beta serine 9 phosphorylation and decrease of CMTM6 expression. In Capan-1 mouse xenograft model, AZD1775 plus AZD0156 (ATM inhibitor) treatment reduced tumor growth and downregulated tumor expression of programmed cell death ligand 1, CMTM6, CD163, and CXCR2, all of which contribute to tumor immune evasion. Conclusion Dual blockade of WEE1 and ATM might be a potential therapeutic strategy for PC. Taken together, our results support further clinical development of DDR-targeting strategies for PC.

Additive Value of B-Type Natriuretic Peptide on Rest 201Tl-Dipyridamole Stress 99mTc-Sestamibi Gated Myocardial SPECT in Patients with Normal Left Ventricular Systolic Function

We evaluated whether BNP has additive value to SPECT in patients with normal left ventricular (LV) systolic function. Data from 224 consecutive patients who underwent rest 201Tl-dipyridamole stress 99mTc-sestamibi gated SPECT and coronary angiography due to chest pain were analyzed. Patients with true positive SPECT showed significant higher BNP level than those with false positive defect (38.5 (19.0–79.8) versus 19.0 (9.3–35.8), P = .01). Patients with true negative SPECT also showed significantly lower BNP level than those with false negative SPECT (39.0 (23.0–77.0) versus 22.0 (15.0–43.0), P = .002). In multivariate analyses, elevated BNP level (using a cut-off value of 23.0 pg/mL) was the strongest and independent predictor of CAD in overall patients (OR 2.75, 95% CI: 1.50–5.023, P = .001) and patients with positive SPECT (OR 3.34, 95% CI: 1.51–7.37, P = .003). The area under the receiver-operating characteristic curve for CAD in overall patients and patients with positive SPECT was 0.673 (95% CI: 0.603–0.743, P < .001) and 0.694 (95% CI: 0.602–0.786, P < .001), respectively. This study suggests that BNP level has additive diagnostic value to SPECT findings in predicting CAD in patients with normal LV systolic function