Multi-scale, whole-system models of liver metabolic adaptation to fat and sugar in non-alcoholic fatty liver disease

Non-alcoholic fatty liver disease (NAFLD) is a serious public health issue associated with high fat, high sugar diets. However, the molecular mechanisms mediating NAFLD pathogenesis are only partially understood. Here we adopt an iterative multi-scale, systems biology approach coupled to in vitro experimentation to investigate the roles of sugar and fat metabolism in NAFLD pathogenesis. The use of fructose as a sweetening agent is controversial; to explore this, we developed a predictive model of human monosaccharide transport, signalling and metabolism. The resulting quantitative model comprising a kinetic model describing monosaccharide transport and insulin signalling integrated with a hepatocyte-specific genome-scale metabolic network (GSMN). Differential kinetics for the utilisation of glucose and fructose were predicted, but the resultant triacylglycerol production was predicted to be similar for monosaccharides; these predictions were verified by in vitro data. The role of physiological adaptation to lipid overload was explored through the comprehensive reconstruction of the peroxisome proliferator activated receptor alpha (PPARα) regulome integrated with a hepatocyte-specific GSMN. The resulting qualitative model reproduced metabolic responses to increased fatty acid levels and mimicked lipid loading in vitro. The model predicted that activation of PPARα by lipids produces a biphasic response, which initially exacerbates steatosis. Our data support the evidence that it is the quantity of sugar rather than the type that is critical in driving the steatotic response. Furthermore, we predict PPARα-mediated adaptations to hepatic lipid overload, shedding light on potential challenges for the use of PPARα agonists to treat NAFLD

Application of In Vivo Induced Antigen Technology (IVIAT) to Bacillus anthracis

In vivo induced antigen technology (IVIAT) is an immuno-screening technique that identifies bacterial antigens expressed during infection and not during standard in vitro culturing conditions. We applied IVIAT to Bacillus anthracis and identified PagA, seven members of a N-acetylmuramoyl-L-alanine amidase autolysin family, three P60 family lipoproteins, two transporters, spore cortex lytic protein SleB, a penicillin binding protein, a putative prophage holin, respiratory nitrate reductase NarG, and three proteins of unknown function. Using quantitative real-time PCR comparing RNA isolated from in vitro cultured B. anthracis to RNA isolated from BALB/c mice infected with virulent Ames strain B. anthracis, we confirmed induced expression in vivo for a subset of B. anthracis genes identified by IVIAT, including L-alanine amidases BA3767, BA4073, and amiA (pXO2-42); the bacteriophage holin gene BA4074; and pagA (pXO1-110). The exogenous addition of two purified putative autolysins identified by IVIAT, N-acetylmuramoyl-L-alanine amidases BA0485 and BA2446, to vegetative B. anthracis cell suspensions induced a species-specific change in bacterial morphology and reduction in viable bacterial cells. Many of the proteins identified in our screen are predicted to affect peptidoglycan re-modeling, and our results support significant cell wall structural remodeling activity during B. anthracis infection. Identification of L-alanine amidases with B. anthracis specificity may suggest new potential therapeutic targets

Small Molecule Control of Virulence Gene Expression in Francisella tularensis

In Francisella tularensis, the SspA protein family members MglA and SspA form a complex that associates with RNA polymerase (RNAP) to positively control the expression of virulence genes critical for the intramacrophage growth and survival of the organism. Although the association of the MglA-SspA complex with RNAP is evidently central to its role in controlling gene expression, the molecular details of how MglA and SspA exert their effects are not known. Here we show that in the live vaccine strain of F. tularensis (LVS), the MglA-SspA complex works in concert with a putative DNA-binding protein we have called PigR, together with the alarmone guanosine tetraphosphate (ppGpp), to regulate the expression of target genes. In particular, we present evidence that MglA, SspA, PigR and ppGpp regulate expression of the same set of genes, and show that mglA, sspA, pigR and ppGpp null mutants exhibit similar intramacrophage growth defects and are strongly attenuated for virulence in mice. We show further that PigR interacts directly with the MglA-SspA complex, suggesting that the central role of the MglA and SspA proteins in the control of virulence gene expression is to serve as a target for a transcription activator. Finally, we present evidence that ppGpp exerts its effects by promoting the interaction between PigR and the RNAP-associated MglA-SspA complex. Through its responsiveness to ppGpp, the contact between PigR and the MglA-SspA complex allows the integration of nutritional cues into the regulatory network governing virulence gene expression

Predictors of relapse and evaluation of the role of postoperative radiation therapy in a modern series of patients with surgically resected stage III (N2) non–small cell lung cancer

Purpose: For patients with stage III (N2) non–small cell lung cancer (NSCLC) treated with surgical resection, postoperative chemotherapy improves overall survival (OS), but the role of postoperative radiation therapy (PORT) is controversial. The purpose of this study was to evaluate risk factors for local-regional recurrence and to evaluate the impact of PORT on local-regional control (LRC) and OS in a modern series of patients with surgically resected stage III (N2) NSCLC. Methods and materials: A retrospective review was performed of patients with Stage III (N2) NSCLC who underwent curative intent resection at our institution between February 1999 and January 2012. OS, LRC, and metastasis-free survival were estimated from the date of surgery using the Kaplan Meier method. Results: A total of 71 patients were included in the study. Patient median age was 63 years. Histology was adenocarcinoma in 69% of patients. Pretreatment positron emission tomography/computed tomography staging was performed for 90% of patients, and preoperative chemotherapy was administered in 23%. The rate of R0 resection was 96%. Forty-one patients (58%) received PORT and the median PORT dose was 50 Gy (range, 41.4-60 Gy). The median follow-up time for living patients was 5.0 years. Five-year OS for all patients was 66%. OS at 5 years for patients who received PORT compared with patients who did not receive PORT was 71% versus 60%, respectively (hazard ratio [HR], 0.61; 95% CI, 0.30-1.44; P = .28). LRC at 5 years for patients who received PORT compared with patients who did not receive PORT was 89% versus 76%, respectively (HR, 0.44; 95% CI, 0.13-1.45; P = .17). Factors associated with decreased LRC were male sex (P = .011) and primary tumor (pT) stage (pT3/4 vs. pT1/2, P = .006). Metastasis-free survival at 5 years for patients who received PORT compared with those who did not receive PORT was 62% versus 63%, respectively (HR, 1.07; 95% CI, 0.51-2.40; P = .86). Conclusions: In this modern series of patients with resected stage III (N2) NSCLC, patients who received PORT had higher rates of OS and LRC, but these differences were not statistically significant

Long-term Clinical Outcomes and Safety Profile of SBRT for Centrally Located NSCLC

Purpose: Previous studies suggest that stereotactic body radiation therapy (SBRT) is associated with higher toxicity rates for central lung tumors relative to peripheral tumors when using 3 fraction SBRT. The initial results from Radiation Therapy Oncology Group study 0813 suggest a safe toxicity profile of SBRT administered in 5 fractions for central non-small cell lung cancer (NSCLC). We reviewed our institutional data to evaluate the safety and efficacy of SBRT for central NSCLC. Methods and materials: We reviewed our prospectively collected SBRT database for patients with central NSCLC who received SBRT between 2008 and 2014. The most frequent dose and fractionations were 50 Gy in 5 fractions (59%) and 48 Gy in 4 fraction (30%). Local control (LC), regional control, metastasis-free survival, and overall survival were calculated using Kaplan-Meier estimates. The National Cancer Institute Common Terminal Criteria for Adverse Events were used for toxicity grading. Results: A total of 110 central lung tumors in 103 patients were included. The median age was 74 years (range, 40-95 years), and the median follow-up time of living patients was 50 months. The mean tumor size was 20 mm (range, 5-70 mm). The 5 year rate of LC, regional control, and distant control was 89%, 77%, and 82%, respectively. The median and 5-year overall survival were 3.5 years and 35%, respectively. No treatment variables were associated with tumor control or other clinical outcomes. A single patient experienced grade 3 radiation pneumonitis (0.97%). The rate of late toxicity grade ≥3 was 9.7% (grade 3, 7.7%; grade 4, 0.97%; grade 5, 0.97%) and included pneumonitis (3.9%), bronchial necrosis (2.9%), myocardial dysfunction (1.9%), and worsening heart failure (0.97%). Conclusions: SBRT for central NSCLC provides high rates of LC. Despite excellent LC, patients remain at risk for regional and distant failure. The rate of grade 3 pneumonitis was consistent with that of prior reports. We observed low rates of grade 4-5 toxicity potentially attributable to SBRT. Our results contribute to the growing body of data in support of the safety of SBRT for central NSCLC

Standardizing Care of Neuro-oncology Patients Using a Customized Electronic Medical Record Toolkit

Objective: To develop and implement a customized toolkit within the electronic medical record (EMR) to standardize care of patients with brain tumors. Patients and Methods: We built a customized structured clinical documentation support toolkit to capture standardized data at office visits. We detail the process by which this toolkit was conceptualized and developed. Toolkit development was a physician-led process to determine a work flow and necessary elements to support best practices as defined by the neuro-oncology clinical team. Results: We have developed in our EMR system a customized work flow for clinical encounters with neuro-oncology patients. In addition to providing a road map for clinical care by our neuro-oncology team, the toolkit is designed to maximize discrete data capture. Several hundred fields of discrete data are captured through the toolkit in the context of our routine office visits. We describe the characteristics of patients seen at our clinic, the adoption of the toolkit, current initiatives supported by the toolkit, and future applications. Conclusion: The EMR can be effectively structured to standardize office visits and improve discrete data capture. This toolkit can be leveraged to support quality improvement and practice-based research initiatives at the point of care in a neuro-oncology practice

Initial experience with intensity modulated proton therapy for intact, clinically localized pancreas cancer: Clinical implementation, dosimetric analysis, acute treatment-related adverse events, and patient-reported outcomes

Purpose: Pencil-beam scanning intensity modulated proton therapy (IMPT) may allow for an improvement in the therapeutic ratio compared with conventional techniques of radiation therapy delivery for pancreatic cancer. The purpose of this study was to describe the clinical implementation of IMPT for intact and clinically localized pancreatic cancer, perform a matched dosimetric comparison with volumetric modulated arc therapy (VMAT), and report acute adverse event (AE) rates and patient-reported outcomes (PROs) of health-related quality of life. Methods and materials: Between July 2016 and March 2017, 13 patients with localized pancreatic cancer underwent concurrent capecitabine or 5-fluorouracil-based chemoradiation therapy (CRT) utilizing IMPT to a dose of 50 Gy (radiobiological effectiveness: 1.1). A VMAT plan was generated for each patient to use for dosimetric comparison. Patients were assessed prospectively for AEs and completed PRO questionnaires utilizing the Functional Assessment of Cancer Therapy-Hepatobiliary at baseline and upon completion of CRT. Results: There was no difference in mean target coverage between IMPT and VMAT (P > .05). IMPT offered significant reductions in dose to organs at risk, including the small bowel, duodenum, stomach, large bowel, liver, and kidneys (P < .05). All patients completed treatment without radiation therapy breaks. The median weight loss during treatment was 1.6 kg (range, 0.1-5.7 kg). No patients experienced grade ≥3 treatment-related AEs. The median Functional Assessment of Cancer Therapy-Hepatobiliary scores prior to versus at the end of CRT were 142 (range, 113-163) versus 136 (range, 107-173; P = .18). Conclusions: Pencil-beam scanning IMPT was feasible and offered significant reductions in radiation exposure to multiple gastrointestinal organs at risk. IMPT was associated with no grade ≥3 gastrointestinal AEs and no change in baseline PROs, but the conclusions are limited due to the patient sample size. Further clinical studies are warranted to evaluate whether these dosimetric advantages translate into clinically meaningful benefits

Intensity Modulated Proton Therapy for Hepatocellular Carcinoma: Initial Clinical Experience

Purpose: Our purpose was to assess the safety and efficacy of intensity modulated proton therapy (IMPT) for the treatment of hepatocellular carcinoma (HCC). Methods and Materials: A retrospective review was conducted on all patients who were treated with IMPT for HCC with curative intent from June 2015 to December 2018. All patients had fiducials placed before treatment. Inverse treatment planning used robust optimization with 2 to 3 beams. The majority of patients were treated in 15 fractions (n = 30, 81%, 52.5-67.5 Gy, relative biological effectiveness), whereas the remainder were treated in 5 fractions (n = 7, 19%, 37.5-50 Gy, relative biological effectiveness). Daily image guidance consisted of orthogonal kilovoltage x-rays and use of a 6° of freedom robotic couch. Outcomes (local control, progression free survival, and overall survival) were determined using Kaplan-Meier methods. Results: Thirty-seven patients were included. The median follow-up for living patients was 21 months (Q1-Q3, 17-30 months). Pretreatment Child-Pugh score was A5-6 in 70% of patients and B7-9 in 30% of patients. Nineteen patients had prior liver directed therapy for HCC before IMPT. Eight patients (22%) required a replan during treatment, most commonly due to inadequate clinical target volume coverage. One patient (3%) experienced a grade 3 acute toxicity (pain) with no recorded grade 4 or 5 toxicities. An increase in Child-Pugh score by ≥ 2 within 3 months of treatment was observed in 6 patients (16%). At 1 year, local control was 94%, intrahepatic control was 54%, progression free survival was 35%, and overall survival was 78%. Conclusions: IMPT is safe and feasible for treatment of HCC