SummaryA two-component signal transduction system composed of a sensor kinase, ResE, and a response regulator, ResD, encoded by resD and resE genes of the res operon (resABCDE), has a regulatory role in both aerobic and anaerobic respiration. In terms of aerobic respiration, resD functions upstream of ctaA, a gene required for haem A biogenesis and hence for the synthesis of haem A-containing cytochrome terminal oxidases. Although ResD is probably a transcription factor, there was no direct evidence that ResD protein, either phosphorylated or unphosphorylated, interacts directly with regulatory regions of ResD-controlled genes. Here, we report the overexpression and purification of ResD and ResE and their role in gene activation. ResD can be phosphorylated by ResE in vitro and is a monomer in solution in either the phosphorylated or unphosphorylated state. The binding activity of ResD to the ctaA promoter was examined by gel shift assays and DNase I footprinting assays. DNase I footprinting showed both unphosphorylated and phosphorylated ResD binding to the ctaA promoter and showed that there are three binding sites (A1, A2 and A3), two (A1 and A2) upstream of the 235 promoter region and one (A3) downstream of the 210 of the promoter. The role of each site in ctaA promoter activity and ResD binding was characterized using deletion analysis, followed by the DNase I footprinting and in vivo transcription assays of promoter±lacZ fusions. Our results showed that the concentration of ResD required to bind at each site is different and that ResD binding at the A1 site is independent of the other two ResD binding sites, but that the concentration of ResD,P required to protect site A2 is reduced when site A3 is present. In vivo transcription assays from promoter± lacZ fusion constructs showed that DNA containing ResD-binding site A2 was essential for promoter activity and that promoter constructs containing both binding sites A2 and A3 were sufficient for full promoter activity.

The Bacillus subtilis ResDE two-component system plays a positive role in global regulation of genes involved in aerobic and anaerobic respiration. ctaA is one of the several genes involved in aerobic respiration that requires ResD for in vivo expression. The ctaAB-divergent promoter regulatory region has three ResD binding sites; A1, A2, and A3. The A2 site is essential for in vivo promoter activity, while binding sites A2 and A3 are required for full ctaA promoter activity. In this study, we demonstrate the role of ResDϳP in the activation of the ctaA promoter using an in vitro transcription system. The results indicate that the ctaA promoter (binding sites A2 and A3) has two transcriptional start sites. Binding site A2 was sufficient for weak transcription of the upstream promoter (Pv) by E The Bacillus subtilis two-component regulatory pair, designated ResD and ResE, has a positive role in global regulation of both aerobic and anaerobic respiration (18,23). The ResDE system is required for transcription of the following genes and operons involved in aerobic respiration; the resABCDE operon (23), encoding proteins similar to those involved in cytochrome c biogenesis (resABC) (6) and ResD-ResE (resDE) (23); the petCBD operon, encoding subunits of the cytochrome bf complex; the ctaBCDEF operon (12), encoding CtaB, which is required for the synthesis of heme O from heme B (ctaB) (25) and structural genes for cytochrome caa 3 (ctaCDEF) (22) and ctaA (23); and a gene required for heme A biogenesis (24, 25) and hence for the synthesis of the heme A-containing terminal cytochrome oxidases aa 3 and caa 3 . Recognition of phenotypic traits shared by resD and ctaA mutants (15) led to a study that revealed that ResD has an essential role in the activation of in vivo expression of the ctaA promoter (23). Phenotypic similarities shared by resD and ctaA mutants, among others, included a sporulation defect and the absence of the heme A-containing terminal oxidases aa 3 and caa 3 . A recent study has shown that either one of these two terminal oxidases is sufficient for sporulation since a qoxABCD (structural genes for aa 3 ) ctaCD (structural genes for caa 3 ) double mutant is sporulation deficient but a single mutant with either mutation is not (28). Thus, the sporulation defect in a resD mutant may be explained by the role of ResD in ctaA and/or ctaB regulation.A direct role for ResD in ctaA promoter activation was suggested in a recent study which showed that there are three ResD binding sites (A1, A2, and A3) in the intercistronic ctaAB promoter region to which either unphosphorylated or phosphorylated ResD binds (29). A1 and A2 are situated upstream of the Ϫ35 promoter region, and A3 is downstream of the Ϫ10 region of the ctaA promoter previously identified (15). Deletion experiments revealed that binding site A1 did not influence the in vivo expression of the ctaA gene (29), suggesting that site A1 may be involved in the regulation of the divergent ctaB promoter, which also requires ResD for expression (12). ctaA-lacZ...

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Purpose: Currently, selection of patients for sequential vs. concurrent chemotherapy/radiation regimens lacks evidentiary support, and it is based on locally-optimal decisions for each step. We aim to optimize the multi-step treatment of head and neck cancer patients and to predict multiple patient survival and toxicity outcomes, and we develop, apply, and evaluate a first application of deep-Q-learning (DQL) and simulation to this problem. Patients and methods: The treatment decision DQL digital twin and the patient's digital twin were created, trained and evaluated on a dataset of 536 oropharyngeal squamous cell carcinoma (OPC) patients with the goal of, respectively, determining the optimal treatment decisions with respect to survival and toxicity metrics, and predicting the outcomes of the optimal treatment on the patient. The models were trained on a subset of 402 patients (split randomly) and evaluated on a separate set of 134 patients. Training and evaluation of the digital twin dyad was completed in August 2020. The dataset includes 3-step sequential treatment decisions and complete relevant history of the patients cohort treated at MD Anderson Cancer Center between 2005 and 2013, with radiomics analysis performed for the segmented primary tumor volumes. Results: On the validation set, 87.09% mean and 90.85% median accuracy in treatment outcome prediction, matching the clinicians' outcomes and improving (predicted) survival rate by +3.73% (95% CI: [-0.75%, +8.96%]), and dysphagia rate by +0.75% (CI: [-4.48%, +6.72%]) when following DQL treatment decisions. Conclusion: Given the prediction accuracy and predicted improvement on medically relevant outcomes yielded by this approach, this digital twin dyad of the patient-physician dynamic treatment problem has the potential of aiding physicians in determining the optimal course of treatment and in assessing its outcomes.