'I'm not a chance taker': A mixed methods exploration of factors affecting prostate cancer treatment decision-making.

OBJECTIVES: African American prostate cancer survivors experience post-treatment decisional regret more often than European Americans, which can lead to negative long-term effects on quality of life. A prominent driver of health-related decision-making is emotion, yet little work has examined the impact emotions may have on decisional regret. The goal of this study was to explore experiences, perceptions, and emotions of prostate cancer survivors in relation to their diagnostic and treatment decision-making processes, prostate cancer treatment, and outcomes. Additionally, we sought to identify factors that might explain differences in prostate cancer outcomes between African and European Americans. DESIGN: This mixed-methods study utilized a convergent parallel design, in which quantitative and qualitative data were collected simultaneously and then integrated to more robustly explain relationships between variables. Survivors were eligible for the study if they had been previously diagnosed with localized prostate cancer and were no more than six months post-treatment. The study was guided by the Risk as Feelings Model, which predicts the relationship between emotion and cognition in high-risk decision-making. RESULTS: No men experienced decisional regret following treatment, even if they experienced side effects. While all men reported being surprised about their prostate cancer diagnosis, strong negative emotions were more common among men under 65. Family support and spirituality appeared to mitigate negative emotions. Perceived authenticity of provider communication was the most influential mediator in men's decision-making and positive perceptions of their outcomes. CONCLUSIONS: To mitigate the impact emotional responses have on decision-making and post-treatment regret, providers should explore alternate therapies (e.g. counseling for men diagnosed with prostate cancer at a young age) and include family members in prostate cancer treatment discussions. Most importantly, providers should be aware of the importance of quality communication on men's cognitive and emotional processes and their perceptions of treatment outcomes.

Disrupting Fluorescence by Mutagenesis in a Green Fluorescent Fatty Acid Binding Protein from a Marine Eel.

Biofluorescence has been found to be an increasingly widespread phenomenon in the ocean. The reclusive Caribbean chlopsid eel, Kaupichthys hyoproroides displays bright green fluorescence in its native marine environment. We have previously shown the fluorescence to be attributed to a fluorescent fatty acid-binding protein, Chlopsid FP, part of a larger family of fluorescent fatty acid-binding proteins, including the homologous UnaG. All require the addition of exogenous bilirubin for fluorescence. Here, we report the generation of a series of point mutants, and deletions that result in the quenching of fluorescence in Chlopsid FP. In addition, we report the binding constants of bilirubin to Chlopsid FP and mutants, measured by fluorescence titration. This study provides key insights into the potential mechanism of fluorescence in this class of fluorescent fatty acid-binding proteins.

Complete Response to Dual Immunotherapy in a Young Adult with Metastatic Alveolar Soft Part Sarcoma Enabled by a Drug Recovery Program in a Community Practice.

Alveolar soft part sarcoma (ASPS) is an extremely rare tumor that frequently occurs in adolescent and young adults (AYA). Survival is poor for patients with metastatic and/or relapsed disease not amenable to local control, and limited therapeutic options are available. A major barrier to cancer care in the United States AYA population is lack of access to coordinated care and appropriate therapies for those who lack insurance or who are underinsured. We report a 25-year-old unemployed, uninsured, single mother who presented with a 12.8 × 21 cm soft tissue thigh mass with heterogeneous avidity, max standardized uptake value of 9, with metastatic disease to the ipsilateral inguinal lymph nodes and to the bilateral lungs. After local control of the primary mass was obtained, a recently developed, comprehensive drug replacement program (DRP) was used to gain access to nivolumab, and after frank progression was noted, ipilimumab was added every 6 weeks. No biomarkers associated with response to immunotherapy were identified. After four cycles, a complete response was observed and patient remains disease free 36 months after beginning dual immunotherapy treatment. We obtained immunotherapy agents through a DRP and describe the development and the utility of this program in the community setting. Our report highlights both first documented sustained complete response to sequenced immunotherapy in an AYA with ASPS as well as a comprehensive DRP, which enabled access to therapy for our patient.

Resolving dual binding conformations of cellulosome cohesin-dockerin complexes using single-molecule force spectroscopy.

Receptor-ligand pairs are ordinarily thought to interact through a lock and key mechanism, where a unique molecular conformation is formed upon binding. Contrary to this paradigm, cellulosomal cohesin-dockerin (Coh-Doc) pairs are believed to interact through redundant dual binding modes consisting of two distinct conformations. Here, we combined site-directed mutagenesis and single-molecule force spectroscopy (SMFS) to study the unbinding of Coh:Doc complexes under force. We designed Doc mutations to knock out each binding mode, and compared their single-molecule unfolding patterns as they were dissociated from Coh using an atomic force microscope (AFM) cantilever. Although average bulk measurements were unable to resolve the differences in Doc binding modes due to the similarity of the interactions, with a single-molecule method we were able to discriminate the two modes based on distinct differences in their mechanical properties. We conclude that under native conditions wild-type Doc from Clostridium thermocellum exocellulase Cel48S populates both binding modes with similar probabilities. Given the vast number of Doc domains with predicted dual binding modes across multiple bacterial species, our approach opens up new possibilities for understanding assembly and catalytic properties of a broad range of multi-enzyme complexes.

Insights into a type III cohesin-dockerin recognition interface from the cellulose-degrading bacterium Ruminococcus flavefaciens.

Cellulosomes are large multicomponent cellulose-degrading assemblies found on the surfaces of cellulolytic microorganisms. Often containing hundreds of components, the self-assembly of cellulosomes is mediated by the ultra-high-affinity cohesin-dockerin interaction, which allows them to adopt the complex architectures necessary for degrading recalcitrant cellulose. Better understanding of how the cellulosome assembles and functions and what kinds of structures it adopts will further effort to develop industrial applications of cellulosome components, including their use in bioenergy production. Ruminococcus flavefaciens is a well-studied anaerobic cellulolytic bacteria found in the intestinal tracts of ruminants and other herbivores. Key to cellulosomal self-assembly in this bacterium is the dockerin ScaADoc, found on the non-catalytic structural subunit scaffoldin ScaA, which is responsible for assembling arrays of cellulose-degrading enzymes. This work expands on previous efforts by conducting a series of binding studies on ScaADoc constructs that contain mutations in their cohesin recognition interface, in order to identify which residues play important roles in binding. Molecular dynamics simulations were employed to gain insight into the structural basis for our findings. A specific residue pair in the first helix of ScaADoc, as well as a glutamate near the C-terminus, was identified to be essential for cohesin binding. By advancing our understanding of the cohesin binding of ScaADoc, this study serves as a foundation for future work to more fully understand the structural basis of cellulosome assembly in R. flavefaciens.

Ultrastable cellulosome-adhesion complex tightens under load.

Challenging environments have guided nature in the development of ultrastable protein complexes. Specialized bacteria produce discrete multi-component protein networks called cellulosomes to effectively digest lignocellulosic biomass. While network assembly is enabled by protein interactions with commonplace affinities, we show that certain cellulosomal ligand-receptor interactions exhibit extreme resistance to applied force. Here, we characterize the ligand-receptor complex responsible for substrate anchoring in the Ruminococcus flavefaciens cellulosome using single-molecule force spectroscopy and steered molecular dynamics simulations. The complex withstands forces of 600-750 pN, making it one of the strongest bimolecular interactions reported, equivalent to half the mechanical strength of a covalent bond. Our findings demonstrate force activation and inter-domain stabilization of the complex, and suggest that certain network components serve as mechanical effectors for maintaining network integrity. This detailed understanding of cellulosomal network components may help in the development of biocatalysts for production of fuels and chemicals from renewable plant-derived biomass.

Elaborate cellulosome architecture of Acetivibrio cellulolyticus revealed by selective screening of cohesin-dockerin interactions.

Cellulosic waste represents a significant and underutilized carbon source for the biofuel industry. Owing to the recalcitrance of crystalline cellulose to enzymatic degradation, it is necessary to design economical methods of liberating the fermentable sugars required for bioethanol production. One route towards unlocking the potential of cellulosic waste lies in a highly complex class of molecular machines, the cellulosomes. Secreted mainly by anaerobic bacteria, cellulosomes are structurally diverse, cell surface-bound protein assemblies that can contain dozens of catalytic components. The key feature of the cellulosome is its modularity, facilitated by the ultra-high affinity cohesin-dockerin interaction. Due to the enormous number of cohesin and dockerin modules found in a typical cellulolytic organism, a major bottleneck in understanding the biology of cellulosomics is the purification of each cohesin- and dockerin-containing component, prior to analyses of their interaction. As opposed to previous approaches, the present study utilized proteins contained in unpurified whole-cell extracts. This strategy was made possible due to an experimental design that allowed for the relevant proteins to be "purified" via targeted affinity interactions as a function of the binding assay. The approach thus represents a new strategy, appropriate for future medium- to high-throughput screening of whole genomes, to determine the interactions between cohesins and dockerins. We have selected the cellulosome of Acetivibrio cellulolyticus for this work due to its exceptionally complex cellulosome systems and intriguing diversity of its cellulosomal modular components. Containing 41 cohesins and 143 dockerins, A. cellulolyticus has one of the largest number of potential cohesin-dockerin interactions of any organism, and contains unusual and novel cellulosomal features. We have surveyed a representative library of cohesin and dockerin modules spanning the cellulosome's total cohesin and dockerin sequence diversity, emphasizing the testing of unusual and previously-unknown protein modules. The screen revealed several novel cell-bound cellulosome architectures, thus expanding on those previously known, as well as soluble cellulose systems that are not bound to the bacterial cell surface. This study sets the stage for screening the entire complement of cellulosomal components from A. cellulolyticus and other organisms with large cellulosome systems. The knowledge gained by such efforts brings us closer to understanding the exceptional catalytic abilities of cellulosomes and will allow the use of novel cellulosomal components in artificial assemblies and in enzyme cocktails for sustainable energy-related research programs.

Single-molecule dissection of the high-affinity cohesin-dockerin complex.

Cellulose-degrading enzyme systems are of significant interest from both a scientific and technological perspective due to the diversity of cellulase families, their unique assembly and substrate binding mechanisms, and their potential applications in several key industrial sectors, notably cellulose hydrolysis for second-generation biofuel production. Particularly fascinating are cellulosomes, the multimodular extracellular complexes produced by numerous anaerobic bacteria. Using single-molecule force spectroscopy, we analyzed the mechanical stability of the intermolecular interfaces between the cohesin and the dockerin modules responsible for self-assembly of the cellulosomal components into the multienzyme complex. The observed cohesin-dockerin rupture forces (>120 pN) are among the highest reported for a receptor-ligand system to date. Using an atomic force microscope protocol that quantified single-molecule binding activity, we observed force-induced dissociation of calcium ions from the duplicated loop-helix F-hand motif located within the dockerin module, which in the presence of EDTA resulted in loss of affinity to the cohesin partner. A cohesin amino acid mutation (D39A) that eliminated hydrogen bonding with the dockerin's critically conserved serine residues reduced the observed rupture forces. Consequently, no calcium loss occurred and dockerin activity was maintained throughout multiple forced dissociation events. These results offer insights at the single-molecule level into the stability and folding of an exquisite class of high-affinity protein-protein interactions that dictate fabrication and architecture of cellulose-degrading molecular machines.

Age-related disparities in the use of radiotherapy for treatment of localized soft tissue sarcoma.

BACKGROUND: Many elderly patients with cancer experience increased cancer-related morbidity and mortality compared with younger patients. In soft tissue sarcoma, adjuvant radiotherapy is an integral part of definitive therapy for limb preservation. The authors of this report hypothesized that age-related disparities exist in the use of radiation. METHODS: Surveillance, Epidemiology, and End Results (SEER) data were used to conduct a retrospective cohort study among patients aged ≥ 25 years who were diagnosed from 1998 to 2004 with nonmetastatic, biopsy-proven, high-grade soft tissue sarcoma of the extremities and underwent a limb-sparing procedure. Patients were stratified according to age (ages < 50 years, 50-70 years, and > 70 years). Logistic regression was used to determine the association between age and the receipt of radiotherapy adjusting for histology, tumor location, tumor size, surgery, sex, race, and marital status. A Cox proportional hazards model was used to compare disease-specific and all-cause mortality. RESULTS: Among 1354 eligible patients; 37.1% were aged > 70 years, 44.3% were women, and 84.4% were Caucasian. Although 73.8% of the cohort received radiotherapy, receipt decreased from 78.2% among patients aged < 50 years to 69.6% among patients aged >70 years (test for trend; P = .006). After adjusting for demographic and tumor factors, older patients remained less likely to receive radiotherapy (odds ratio, 0.66; 95% confidence interval, 0.47-0.92) and more likely to experience disease-specific death (hazard ratio, 2.4; 95% confidence interval, 1.4-4.1) compared with the youngest group. CONCLUSIONS: Older adults appeared to be less likely to receive definitive therapy for soft tissue sarcoma of the extremities. In the absence of clinical trials and treatment guidelines tailored to this population, under treatment may disadvantage elderly patients, who have increased cancer-related morbidity and mortality.

Bipartite tetracysteine display requires site flexibility for ReAsH coordination.

Flexibility required: We designed intramolecular bipartite tetracysteine sites in loops of p53 and the beta-sheets of EmGFP. We found that ReAsH binding preferentially favors tetracysteine sites with flexible geometries such as loops; flexibility was assessed by comparing Calpha B-factor values. This information is important for directing successful bipartite tetracysteine site designs.

Surveying polypeptide and protein domain conformation and association with FlAsH and ReAsH.

Recombinant polypeptides and protein domains containing two cysteine pairs located distal in primary sequence but proximal in the native folded or assembled state are labeled selectively in vitro and in mammalian cells using the profluorescent biarsenical reagents FlAsH-EDT2 and ReAsH-EDT2. This strategy, termed bipartite tetracysteine display, enables the detection of protein-protein interactions and alternative protein conformations in live cells. As proof of principle, we show that the equilibrium stability and fluorescence intensity of polypeptide-biarsenical complexes correlates with the thermodynamic stability of the protein fold or assembly. Destabilized protein variants form less stable and less bright biarsenical complexes, which allows discrimination of live cells expressing folded polypeptide and protein domains from those containing disruptive point mutations. Bipartite tetracysteine display may provide a means to detect early protein misfolding events associated with Alzheimer's disease, Parkinson's disease and cystic fibrosis; it may also enable high-throughput screening of compounds that stabilize discrete protein folds.

Third-generation chemotherapy agents in the treatment of advanced non-small cell lung cancer: a meta-analysis.

PURPOSE: To estimate the efficacy of third-generation (3G) chemotherapy agents (paclitaxel, docetaxel, gemcitabine, vinorelbine, and irinotecan) on response and survival in stage IIIB/IV non-small cell lung cancer (NSCLC). METHODS: A meta-analysis was performed using trials identified through MEDLINE. Results on tumor response and survival were collected from randomized trials comparing 3G monotherapy versus best supportive care (BSC), 3G monotherapy versus second-generation (2G) platinum-based regimens, and 3G platinum-based regimens versus 2G platinum-based regimens. RESULTS: Of the 2480 citations screened, 20 randomized controlled trials fulfilled the inclusion and exclusion criteria, and 19 trials were used in the analyses. The data from two, three-arm trials were used in two different comparisons. Five trials (n = 1029 patients) compared 3G monotherapy with BSC. The summary risk difference (RD) for 1-year survival favored 3G agents by 7% (95% confidence interval [CI]: 2%, 12%). Four trials (n = 871 patients) compared treatment with 3G monotherapy versus 2G platinum-based regimens. The response RD was -6% (95% CI: -11%, 0%), and the 1-year survival rate RD was 3% (95% CI: -3%, 10%), suggesting that despite a slightly higher response rate for 2G platinum-based regimens relative to 3G monotherapy, there is equivalency in survival. Twelve trials (n = 3995) compared 3G versus 2G platinum-based regimens. The RD for response was 12% (95% CI: 10%, 15%). A RD for 1-year was not calculated, because of heterogeneity among the trials. A subset analysis of 3G versus 2G platinum-based doublets revealed a 1-year survival-rate RD of 6% (95% CI: 2%, 10%), favoring 3G platinum-based regimens without evidence of heterogeneity. CONCLUSIONS: 3G agents have been a significant advance in the treatment of NSCLC.

Current and evolving treatment options for limited stage small cell lung cancer.

PURPOSE OF REVIEW: About one-third of small cell lung cancer cases are classified as limited stage. Trials have attempted to establish the most effective, least toxic regimen of combined modality treatment. Recently, issues like the role of the positron emission tomography scan, elderly patient management and the timing and delivery of radiotherapy have been addressed. Several targeted agents have also been evaluated. This review will highlight the data that have greatly impacted on the standard of limited stage small cell lung cancer care. RECENT FINDINGS: Trials have concluded that small cell lung cancer is fluorodeoxy-D-glucose avid and that positron emission tomography has potential for utility in staging and radiation therapy planning, though it is not recommended. Recent trials confirm no benefit to adding chemotherapeutic agents to standard cisplatin and etoposide. To date, all targeted therapies have failed to show impressive results. Two analyses of outcomes in elderly patients argue that combined modality therapy should be considered, with patients carefully monitored. Two meta-analyses demonstrate thoracic radiotherapy should be delivered, with increased dose and schedule intensity. SUMMARY: Current data demonstrate that combined modality therapy with early administration of thoracic radiotherapy remains the care standard in limited stage small cell lung cancer care. Cisplatin and etoposide continue to be the chosen cytotoxic agents. Targeted therapies and advances in the radiotherapy technological aspects represent opportunities for improved outcomes in the future management of this aggressive disease.

Systematic review evaluating the timing of thoracic radiation therapy in combined modality therapy for limited-stage small-cell lung cancer.

PURPOSE: We employed meta-analytic techniques to evaluate early (E) versus late (L) timing of thoracic radiation therapy (RT) in limited-stage small-cell lung cancer (LS-SCLC). In addition, we assessed the impact of radiation fractionation and chemotherapeutic regimen on timing. METHODS: Randomized trials published after 1985 addressing timing of RT relative to chemotherapy in LS-SCLC were included. Trials were analyzed by risk ratio (RR), risk difference, and number-needed-to-treat methods. RESULTS: Overall survival (OS) RRs for all studies were 1.17 at 2 years (95% CI, 1.02 to 1.35; P = .03) and 1.13 at 3 years (95% CI, 0.92 to 1.39; P = .2), indicating a significantly increased 2-year survival for ERT versus LRT patients and suggestive of a similar trend at 3 years. Subset analysis of studies using hyperfractionated RT revealed OS RR for ERT versus LRT of 1.44 (95% CI, 1.17 to 1.77; P = .001) and 1.39 (95% CI, 1.02 to 1.90; P = .04) at 2 and 3 years, respectively, indicating a survival benefit of ERT versus LRT. Studies using once-daily fractionation showed no difference in 2- and 3-year OS RRs for ERT compared with LRT. Studies using platinum-based chemotherapy had OS RRs of 1.30 (95% CI, 1.10 to 1.53; P = .002) and 1.35 (95% CI, 1.07 to 1.70; P = .01) at 2 and 3 years, respectively, favoring ERT. Studies using nonplatinum-based chemotherapy regimens had nonsignificant differences in OS. CONCLUSION: A small but significant improvement in 2-year OS for ERT versus LRT in LS-SCLC was observed, similar to the benefit of adding RT to chemotherapy or prophylactic cranial irradiation. A greater difference was evident for hyperfractionated RT and platinum-based chemotherapy.