Although the denaturant-induced unfolding transition of cytochrome c was initially thought to be a cooperative process, recent spectroscopic studies have shown deviations from two-state behavior consistent with accumulation of an equilibrium intermediate. However, little is known about the structural and thermodynamic properties of this state, and whether it is stabilized by the presence of non-native heme ligands. We monitored the reversible denaturant-induced unfolding equilibrium of oxidized horse cytochrome c using various spectroscopic probes, including fluorescence, near and far-UV CD, heme absorbance bands in the Soret, visible and near-IR regions of the spectrum, as well as 2D NMR. Global fitting techniques were used for a quantitative interpretation of the results in terms of a three-state model, which enabled us to determine the intrinsic spectroscopic properties of the intermediate. A well-populated intermediate was observed in equilibrium experiments at pH 5 using either guanidine-HCl or urea as a denaturant, both for wild-type cytochrome c as well as an H33N mutant chosen to prevent formation of non-native Hisheme ligation. For a more detailed structural characterization of the intermediate, we used 2D 1 H-15 N correlation spectroscopy to follow the changes in peak intensity for individual backbone amide groups. The equilibrium state observed in our optical and NMR studies contains many native-like structural features, including a well-structured a-helical subdomain, a short Trp59-heme distance and solvent-shielded heme environment, but lacks the native Met80 sulfur-iron linkage and shows major perturbations in side-chain packing and other tertiary interactions. These structural properties are reminiscent of the A-state of cytochrome c, a compact denatured form found under acidic high-salt conditions, as well as a kinetic intermediate populated at a late stage of folding. The denaturant-induced intermediate also resembles alkaline forms of cytochrome c with altered heme ligation, suggesting that disruption of the native methionine ligand favors accumulation of structurally analogous states both in the presence and absence of non-native ligands.

What’s known on the subject? and What does the study add? Prior to publication of our paper, evidence had suggested elevated activity of the mTOR pathway in urothelial carcinoma. Separate studies had in addition demonstrated that mTOR inhibition sensitized certain cancer cells to cisplatin therapy. We therefore evaluated the efficacy of mTOR inhibition using in vitro and in vivo models of bladder cancer, and investigated whether mTOR inhibition accentuates the response of bladder cancer cells to cisplatin, which is currently the most effective systemic treatment for metastatic bladder cancer. We found that mTOR blockade inhibits urothelial carcinoma cell proliferation, and enhances the therapeutic efficacy of cisplatin. Our data support a functional relevance for the mTOR signaling pathway in bladder cancer, and provide evidence to suggest the potential for mTOR inhibitors to be used in combination with cisplatin‐based chemotherapy regimens. OBJECTIVE • To investigate whether mammalian target of rapamycin (mTOR) inhibition by rapamycin is therapeutically efficacious in combination with cisplatin for bladder cancer. MATERIALS AND METHODS • Using a panel of human urothelial carcinoma cell lines, we determined the effect of rapamycin on cell viability, cell‐cycle progression, signalling and apoptosis. • The effect of mTOR inhibition on chemosensitivity was investigated by treating cells with rapamycin, alone, or with cisplatin. • The effect of rapamycin or cisplatin treatment was assessed in xenograft mice inoculated with urothelial carcinoma cells. • Expression of p‐mTOR in human bladder cancer specimens was assessed using a tissue microarray. RESULTS • Treatment with rapamycin significantly decreased cell viability in UMUC3 (P= 0.004) and 253J (P < 0.001) cells. It induced arrest in the G0‐G1 phase and decreased activation of p‐mTOR and its downstream effector, p‐S6K, in both cell lines. • Treatment with rapamycin increased the ability of cisplatin to inhibit cell viability in UMUC3 (P= 0.002) and 253J (P= 0.03) cells. No evidence for apoptosis induction was noted after treatment with rapamycin alone. • Mouse xenografts of UMUC3 cells revealed that rapamycin significantly prolonged survival and enhanced the therapeutic efficacy of cisplatin. • In patient urothelial carcinoma specimens, p‐mTOR expression was increased in cancer vs non‐tumour bladder tissue in 65/203 (32.0%) tumours. CONCLUSIONS • mTOR blockade inhibits urothelial carcinoma cell proliferation and enhances the effectiveness of cisplatin. • Suppression of the mTOR pathway has the potential to be a therapeutic target in bladder cancer for selected patients.

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Bayesian neural networks (BNNs) have drawn extensive interest due to the unique probabilistic representation framework. However, Bayesian neural networks have limited publicized deployments because of the relatively poor model performance in real-world applications. In this paper, we argue that the randomness of sampling in Bayesian neural networks causes errors in the updating of model parameters during training and some sampled models with poor performance in testing. To solve this, we propose to train Bayesian neural networks with Adversarial Distribution as a theoretical solution. To avoid the difficulty of calculating Adversarial Distribution analytically, we further present the Adversarial Sampling method as an approximation in practice. We conduct extensive experiments with multiple network structures on different datasets, e.g., CIFAR-10 and CIFAR-100. Experimental results validate the correctness of the theoretical analysis and the effectiveness of the Adversarial Sampling on improving model performance. Additionally, models trained with Adversarial Sampling still keep their ability to model uncertainties and perform better when predictions are retained according to the uncertainties, which further verifies the generality of the Adversarial Sampling approach.

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