Endoscopic and imaging outcomes of PD-1 therapy in localised dMMR colorectal cancer.

BACKGROUND: Neoadjuvant immune checkpoint blockade (IO) is emerging as a therapeutic option for patients with deficient mismatch repair (dMMR) colorectal cancer (CRC) given high pathological response rates. The aim of the study was to characterise imaging and endoscopic response to IO. METHODS: A retrospective analysis of patients with localised dMMR CRC that received at least one cycle of neoadjuvant anti-PD-1 therapy was conducted. Endoscopy, imaging, and pathological outcomes were reviewed to determine response to treatment according to standardised criteria. RESULTS: Thirty-eight patients had received IO for the treatment of localised CRC (median eight cycles). Among evaluable cases (n = 31 for endoscopy and n = 34 for imaging), the best endoscopic response was complete response (CR) in 45% of cases, and the best radiographic response was CR in 23% of cases. Imaging CR rate after ≤4 cycles of IO (n = 1) was 6% compared to 44% after >4 IO cycles (n = 7). Among 28 patients with imaging and endoscopy available, a discrepancy in best response was noted in 15 (54%) cases. At a median follow-up of 28.2 months from IO start, 18 patients underwent surgical resection of which 11 (61%) had pathological CR (pCR). Despite pCR or no evidence of progression ≥6 months after completion of IO among non-operatively managed patients, 72% and 42% of patients had non-CR on imaging and endoscopy, respectively. CONCLUSIONS: Discrepancies between imaging and endoscopy are prevalent, and irregularities identified on these modalities can be identified despite pathological remission. Improved clinical response criteria are warranted.

Exploring perception and usage of narrative medicine by physician specialty: a qualitative analysis.

BACKGROUND: Narrative medicine is a well-recognized and respected approach to care. It is now found in medical school curricula and widely implemented in practice. However, there has been no analysis of the perception and usage of narrative medicine across different medical specialties and whether there may be unique recommendations for implementation based upon specialty. The aims of this study were to explore these gaps in research. METHODS: Fifteen senior physicians who specialize in internal medicine, pediatrics, or surgery (5 physicians from each specialty) were interviewed in a semi-structured format about the utilization, benefits, drawbacks (i.e., negative consequences), and roles pertaining to narrative medicine. Qualitative content analysis of each interview was then performed. RESULTS: Three themes emerged from our analysis: roles, practice, and outcomes. Through these themes we examined the importance, utilization, barriers, benefits, and drawbacks of narrative medicine. There was consensus that narrative medicine is an important tool in primary care. Primary care physicians (general internists and general pediatricians) also believed that narrative medicine is not as important for non-primary care providers. However, non-primary care providers (surgeons) generally believed narrative medicine is valuable in their practice as well. Within specialties, providers' choice of language varied when trying to obtain patients' narratives, but choice in when to practice narrative medicine did not differ greatly. Among specialties, there was more variability regarding when to practice narrative medicine and what barriers were present. Primary care physicians primarily described barriers to eliciting a patient's narrative to involve trust and emotional readiness, while surgeons primarily described factors involving logistics and patient data as barriers to obtaining patients' narratives. There was broad agreement among specialties regarding the benefits and drawbacks of narrative medicine. CONCLUSIONS: This study sheds light on the shared and unique beliefs in different specialties about narrative medicine. It prompts important discussion around topics such as the stereotypes physicians may hold about their peers and concerns about time management. These data provide some possible ideas for crafting narrative medicine education specific to specialties as well as future directions of study.

TM1385 from Thermotoga maritima functions as a phosphoglucose isomerase via cis-enediol-based mechanism with active site redundancy.

Phosphoglucose isomerases (PGIs) belong to a class of enzymes that catalyze the reversible isomerization of glucose-6-phosphate to fructose-6-phosphate. PGIs are crucial in glycolysis and gluconeogenesis pathways and proposed as serving additional extracellular functions in eukaryotic organisms. The phosphoglucose isomerase function of TM1385, a previously uncharacterized protein from Thermotoga maritima, was hypothesized based on structural similarity to established PGI crystal structures and computational docking. Kinetic and colorimetric assays combined with 1H nuclear magnetic resonance (NMR) spectroscopy experimentally confirm that TM1385 is a phosphoglucose isomerase (TmPGI). Evidence of solvent exchange in 1H NMR spectra supports that TmPGI isomerization proceeds through a cis-enediol-based mechanism. To determine which amino acid residues are critical for TmPGI catalysis, putative active site residues were mutated with alanine and screened for activity. Results support that E281 is most important for TmPGI formation of the cis-enediol intermediate, and the presence of either H310 or K422 may be required for catalysis, similar to previous observations from homologous PGIs. However, only TmPGI E281A/Q415A and H310A/K422A double mutations abolished activity, suggesting that there are redundant catalytic residues, and Q415 may participate in sugar phosphate isomerization upon E281 mutation. Combined, we propose that TmPGI E281 participates directly in the cis-enediol intermediate step, and either H310 or K422 may facilitate sugar ring opening and closure.

Opportunities to Improve Shared Decision Making in Dialysis Decisions for Older Adults with Life-Limiting Kidney Disease: A Pilot Study.

Background: Lack of awareness about the life-limiting nature of renal failure is a significant barrier to palliative care for older adults with end-stage renal disease. Objective: To train nephrologists to use the best case/worst case (BC/WC) communication tool to improve shared decision making about dialysis initiation for older patients with limited life expectancy. Design: This is a pre-/postinterventional pilot study. Setting/Subjects: There were 16 nephrologists and 30 patients of age 70 years and older with estimated glomerular filtration rate (eGFR) <20 mL/min per 1.73 m2 in outpatient nephrology clinics, in Madison, WI. Measurements: Performance of tool elements, content of communication about dialysis, shared decision making, acceptability of the intervention, decisions to pursue dialysis, and palliative care referrals were measured. Results: Fifteen of 16 nephrologists achieved competence performing the BC/WC tool with standardized patients, executing at least 14 of 19 items. Nine nephrologists met with 30 patients who consented to audio record their clinic visit. Before training, clinic visits focused on laboratory results and preparation for dialysis. After training, nephrologists noted that declining kidney function was "bad news," presented dialysis and "no dialysis" as treatment options, and elicited patient preferences. Observer-measured shared decision-making (OPTION 5) scores improved from a median of 20/100 (interquartile range [IQR] 15-35) before training to 58/100 (IQR 55-65). Patients whose nephrologist used the BC/WC tool were less likely to make a decision to initiate dialysis and were more likely to be referred to palliative care. Conclusions: Nephrologists can learn to use the BC/WC tool with older patients to improve shared decision making about dialysis, which may increase access to palliative care.

Effectiveness of a Question Prompt List Intervention for Older Patients Considering Major Surgery: A Multisite Randomized Clinical Trial.

Importance: Poor preoperative communication can have serious consequences, including unwanted treatment and postoperative conflict. Objective: To compare the effectiveness of a question prompt list (QPL) intervention vs usual care on patient engagement and well-being among older patients considering major surgery. Design, Setting, and Participants: This randomized clinical trial used a stepped-wedge design to randomly assign patients to a QPL intervention (n = 223) or usual care (n = 223) based on the timing of their visit with 1 of 40 surgeons at 5 US study sites. Patients were 60 years or older with at least 1 comorbidity and an oncologic or vascular (cardiac, neurosurgical, or peripheral vascular) problem that could be treated with major surgery. Family members were also enrolled (n = 263). The study dates were June 2016 to November 2018. Data analysis was by intent-to-treat. Interventions: A brochure of 11 questions to ask a surgeon developed by patient and family stakeholders plus an endorsement letter from the surgeon were sent to patients before their outpatient visit. Main Outcomes and Measures: Primary patient engagement outcomes included the number and type of questions asked during the surgical visit and patient-reported Perceived Efficacy in Patient-Physician Interactions scale assessed after the surgical visit. Primary well-being outcomes included (1) the difference between patient's Measure Yourself Concerns and Well-being (MYCaW) scores reported after surgery and scores reported after the surgical visit and (2) treatment-associated regret at 6 to 8 weeks after surgery. Results: Of 1319 patients eligible for participation, 223 were randomized to the QPL intervention and 223 to usual care. Among 446 patients, the mean (SD) age was 71.8 (7.1) years, and 249 (55.8%) were male. On intent-to-treat analysis, there was no significant difference between the QPL intervention and usual care for all patient-reported primary outcomes. The difference in MYCaW scores for family members was greater in usual care (effect estimate, 1.51; 95% CI, 0.28-2.74; P = .008). When the QPL intervention group was restricted to patients with clear evidence they reviewed the QPL, a nonsignificant increase in the effect size was observed for questions about options (odds ratio, 1.88; 95% CI, 0.81-4.35; P = .16), expectations (odds ratio, 1.59; 95% CI, 0.67-3.80; P = .29), and risks (odds ratio, 2.41; 95% CI, 1.04-5.59; P = .04) (nominal α = .01). Conclusions and Relevance: The results of this study were null related to primary patient engagement and well-being outcomes. Changing patient-physician communication may be difficult without addressing clinician communication directly. Trial Registration: ClinicalTrials.gov identifier: NCT02623335.

Tuning micelle dimensions and properties with binary surfactant mixtures.

Detergent micelles are used in many areas of research and technology, in particular, as mimics of the cellular membranes in the purification and biochemical and structural characterization of membrane proteins. Applications of detergent micelles are often hindered by the limited set of properties of commercially available detergents. Mixtures of micelle-forming detergents provide a means to systematically obtain additional micellar properties and expand the repertoire of micelle features available; however, our understanding of the properties of detergent mixtures is still limited. In this study, the shape and size of binary mixtures of seven different detergents commonly used in molecular host-guest systems and membrane protein research were investigated. The data suggests that the detergents form ideally mixed micelles with sizes and shapes different from those of pure individual micelles. For most measurements of size, the mixtures varied linearly with detergent mole fraction and therefore can be calculated from the values of the pure detergents. We propose that properties such as the geometry, size, and surface charge can be systematically and predictably tuned for specific applications.

Structure of the Neisserial outer membrane protein Opa60: loop flexibility essential to receptor recognition and bacterial engulfment.

The structure and dynamics of Opa proteins, which we report herein, are responsible for the receptor-mediated engulfment of Neisseria gonorrheae or Neisseria meningitidis by human cells and can offer deep understanding into the molecular recognition of pathogen-host receptor interactions. Such interactions are vital to understanding bacterial pathogenesis as well as the mechanism of foreign body entry to a human cell, which may provide insights for the development of targeted pharmaceutical delivery systems. The size and dynamics of the extracellular loops of Opa60 required a hybrid refinement approach wherein membrane and distance restraints were used to generate an initial NMR structural ensemble, which was then further refined using molecular dynamics in a DMPC bilayer. The resulting ensemble revealed that the extracellular loops, which bind host receptors, occupy compact conformations, interact with each other weakly, and are dynamic on the nanosecond time scale. We predict that this conformational sampling is critical for enabling diverse Opa loop sequences to engage a common set of receptors.

Solution NMR resonance assignment strategies for ß-barrel membrane proteins.

Membrane proteins in detergent micelles are large and dynamic complexes that present challenges for solution NMR investigations such as spectral overlap and line broadening. In this study, multiple methods are introduced to facilitate resonance assignment of ß-barrel membrane proteins using Opa60 from Neisseria gonorrhoeae as a model system. Opa60 is an eight-stranded ß-barrel with long extracellular loops (∼63% of the protein) that engage host receptors and induce engulfment of the bacterium. The NMR spectra of Opa60 in detergent micelles exhibits significant spectral overlap and resonances corresponding to the loop regions had variable line widths, which interfered with a complete assignment of the protein. To assign the ß-barrel residues, trypsin cleavage was used to remove much of the extracellular loops while preserving the detergent solubilized ß-barrel. The removal of the loop resonances significantly improved the assignment of the Opa60 ß-barrel region (97% of the resonances corresponding to the ß-barrel and periplasmic turns were assigned). For the loop resonance assignments, two strategies were implemented; modulating temperature and synthetic peptides. Lowering the temperature broadened many peaks beyond detection and simplified the spectra to only the most dynamic regions of the loops facilitating 27 loop resonances to be assigned. To further assign functionally important and unstructured regions of the extracellular loops, a synthetic 20 amino acid peptide was synthesized and had nearly complete spectral overlap with the full-length protein allowing 17 loop resonances to be assigned. Collectively, these strategies are effective tools that may accelerate solution NMR structure determination of ß-barrel membrane proteins.

Dependence of micelle size and shape on detergent alkyl chain length and head group.

Micelle-forming detergents provide an amphipathic environment that can mimic lipid bilayers and are important tools for solubilizing membrane proteins for functional and structural investigations in vitro. However, the formation of a soluble protein-detergent complex (PDC) currently relies on empirical screening of detergents, and a stable and functional PDC is often not obtained. To provide a foundation for systematic comparisons between the properties of the detergent micelle and the resulting PDC, a comprehensive set of detergents commonly used for membrane protein studies are systematically investigated. Using small-angle X-ray scattering (SAXS), micelle shapes and sizes are determined for phosphocholines with 10, 12, and 14 alkyl carbons, glucosides with 8, 9, and 10 alkyl carbons, maltosides with 8, 10, and 12 alkyl carbons, and lysophosphatidyl glycerols with 14 and 16 alkyl carbons. The SAXS profiles are well described by two-component ellipsoid models, with an electron rich outer shell corresponding to the detergent head groups and a less electron dense hydrophobic core composed of the alkyl chains. The minor axis of the elliptical micelle core from these models is constrained by the length of the alkyl chain, and increases by 1.2-1.5 Šper carbon addition to the alkyl chain. The major elliptical axis also increases with chain length; however, the ellipticity remains approximately constant for each detergent series. In addition, the aggregation number of these detergents increases by ∼16 monomers per micelle for each alkyl carbon added. The data provide a comprehensive view of the determinants of micelle shape and size and provide a baseline for correlating micelle properties with protein-detergent interactions.

Mixing and matching detergents for membrane protein NMR structure determination.

One major obstacle to membrane protein structure determination is the selection of a detergent micelle that mimics the native lipid bilayer. Currently, detergents are selected by exhaustive screening because the effects of protein-detergent interactions on protein structure are poorly understood. In this study, the structure and dynamics of an integral membrane protein in different detergents is investigated by nuclear magnetic resonance (NMR) and electron paramagnetic resonance (EPR) spectroscopy and small-angle X-ray scattering (SAXS). The results suggest that matching of the micelle dimensions to the protein's hydrophobic surface avoids exchange processes that reduce the completeness of the NMR observations. Based on these dimensions, several mixed micelles were designed that improved the completeness of NMR observations. These findings provide a basis for the rational design of mixed micelles that may advance membrane protein structure determination by NMR.