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Transparent patient-centered communication is essential to providing quality hospice care for patients at the end of life. This study aimed to determine and describe the current state of aid-in-dying policies in California and their effect on hospice nursing in response to narratives about leave-the-room policies presenting professional and moral challenges. In total, 97 hospice program policies were analyzed with a focus on the role of nurses at the bedside and intent to discharge patients who pursue medical aid-in-dying. It is necessary to clarify the important role of hospice nurses who care for terminally ill patients pursuing their legal right to assisted dying. The results of this study underscore the need for improved policy transparency and organizational support to enhance hospice engagement, particularly by nurses, with their patients at the end of life.

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OBJECTIVE: To identify the frequency and nature of care conflict dilemmas that United States long-term care providers encounter, response strategies, and use of ethics resources to assist with dispute resolution. DESIGN: An online cross-sectional survey was distributed to the Society for Post-Acute and Long-Term Care Medicine (AMDA). RESULTS: Two-thirds of participants, primarily medical directors, have rejected surrogate instructions and 71% have managed family conflict. Conflict over treatment decisions and issues interpreting advance directives were frequently reported. Half of facilities lack a formal dispute mediation policy. Only five respondents have called an ethics consult for assistance. CONCLUSION: Ethically tense care conflicts commonly arise in long-term and post-acute care facilities. Few facility procedures incorporate ethics resources into actual practice. Recommendations are made to create actionable policy, increase access to ethics services, and support staff skill development in order to improve the end-of-life care experiences for patients, families, and care facility staff.

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OBJECTIVE: To evaluate the effectiveness of resident-led and faculty-led initiatives for physician wellness after implementation of a resident wellness program. DESIGN: We initiated a wellness curriculum with both resident and faculty-led components in a large academic OB/GYN residency program in October 2020. The curriculum was created and evaluated using the Logic model. Residents were surveyed pre and 8 months postintervention with the Maslach Burnout Inventory (MBI) and the Physician Well-Being Index (PWBI), with activity-related questions added to the second survey. Descriptive statistics, Mann-Whitney test, Chi-square test, and theme analysis were performed as appropriate. SETTING: A large academic OB/GYN residency at Baylor College of Medicine in Houston, Texas PARTICIPANTS: All residents (n = 48) were invited to take part in the surveys. Response rate was 31/48 (65%) pre and 28/48 (58%) postintervention. RESULTS: Residents scored moderate for emotional exhaustion and depersonalization and high for personal accomplishment on both pre and post-MBI surveys. All indices of the PWBI improved over time; however, no significant differences were found in pre and postmeasures. Resident-led activities, which were alternated between individualized time off and group resident socialization, were rated significantly higher than faculty-led activities; 93% (52/56) of respondents rated resident-led activities in their top 2 most helpful initiatives compared to 7% (4/56) who rated faculty-led activities in their top 2 most helpful (p < 0.01) initiatives. Open-ended comments revealed that continued focus on wellness, attention to personal health, and systematic change were the most important ways to improve resident wellness. CONCLUSION: Decreases in burnout were not achieved over an 8-month period with program-level resident-led and faculty-led initiatives. Providing scheduled time for residents to use at their discretion and the continuation of events that encourage socialization are tools that are highest rated by residents to facilitate wellness.

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To evaluate the clinical impact of molecular tumor profiling (MTP) with targeted sequencing panel tests, pediatric patients with extracranial solid tumors were enrolled in a prospective observational cohort study at 12 institutions. In the 345-patient analytical population, median age at diagnosis was 12 years (range 0-27.5); 298 patients (86%) had 1 or more alterations with potential for impact on care. Genomic alterations with diagnostic, prognostic or therapeutic significance were present in 61, 16 and 65% of patients, respectively. After return of the results, impact on care included 17 patients with a clarified diagnostic classification and 240 patients with an MTP result that could be used to select molecularly targeted therapy matched to identified alterations (MTT). Of the 29 patients who received MTT, 24% had an objective response or experienced durable clinical benefit; all but 1 of these patients received targeted therapy matched to a gene fusion. Of the diagnostic variants identified in 209 patients, 77% were gene fusions. MTP with targeted panel tests that includes fusion detection has a substantial clinical impact for young patients with solid tumors.

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PURPOSE: Molecular tumor profiling is becoming a routine part of clinical cancer care, typically involving tumor-only panel testing without matched germline. We hypothesized that integrated germline sequencing could improve clinical interpretation and enhance the identification of germline variants with significant hereditary risks. MATERIALS AND METHODS: Tumors from pediatric patients with high-risk, extracranial solid malignancies were sequenced with a targeted panel of cancer-associated genes. Later, germline DNA was analyzed for a subset of these genes. We performed a post hoc analysis to identify how an integrated analysis of tumor and germline data would improve clinical interpretation. RESULTS: One hundred sixty participants with both tumor-only and germline sequencing reports were eligible for this analysis. Germline sequencing identified 38 pathogenic or likely pathogenic variants among 35 (22%) patients. Twenty-five (66%) of these were included in the tumor sequencing report. The remaining germline pathogenic or likely pathogenic variants were single-nucleotide variants filtered out of tumor-only analysis because of population frequency or copy-number variation masked by additional copy-number changes in the tumor. In tumor-only sequencing, 308 of 434 (71%) single-nucleotide variants reported were present in the germline, including 31% with suggested clinical utility. Finally, we provide further evidence that the variant allele fraction from tumor-only sequencing is insufficient to differentiate somatic from germline events. CONCLUSION: A paired approach to analyzing tumor and germline sequencing data would be expected to improve the efficiency and accuracy of distinguishing somatic mutations and germline variants, thereby facilitating the process of variant curation and therapeutic interpretation for somatic reports, as well as the identification of variants associated with germline cancer predisposition.

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INTRODUCTION: Tissue from pediatric solid tumors is in high demand for use in high-impact research studies, making the allocation of tissue from an anatomic pathology laboratory challenging. We designed, implemented, and assessed an interdepartmental process to optimize tissue allocation of pediatric solid tumors for both clinical care and research. METHODS: Oncologists, pathologists, surgeons, interventional radiologists, pathology technical staff, and clinical research coordinators participated in the workflow design. Procedures were created to address patient identification and consent, prioritization of protocols, electronic communication of requests, tissue preparation, and distribution. Pathologists were surveyed about the value of the new workflow. RESULTS: Over a 5-year period, 644 pediatric solid tumor patients consented to one or more studies requesting archival or fresh tissue. Patients had a variety of tumor types, with many rare and singular diagnoses. Sixty-seven percent of 1768 research requests were fulfilled. Requests for archival tissue were fulfilled at a significantly higher rate than those for fresh tissue (P > .001), and requests from resection specimens were fulfilled at a significantly higher rate than those from biopsies (P > .0001). In an anonymous survey, seven of seven pathologists reported that the process had improved since the introduction of the electronic communication model. CONCLUSIONS: A collaborative and informed model for tissue allocation is successful in distributing archival and fresh tissue for clinical research studies. Our workflows and policies have gained pathologists' approval and streamlined our processes. As clinical and research programs evolve, a thoughtful tissue allocation process will facilitate ongoing research.

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This paper presents an exploratory study of the binding interactions of xenon with the surface of several different proteins in the solution and solid states using both conventional and hyperpolarized (129)Xe NMR. The generation of hyperpolarized (129)Xe by spin exchange optical pumping affords an enhancement by 3-4 orders of magnitude of its NMR signal. As a result, it is possible to observe Xe directly bound to the surface of micromolar quantities of lyophilized protein. The highly sensitive nature of the (129)Xe line shape and chemical shift are used as indicators for the conditions most likely to yield maximal dipolar contact between (129)Xe nuclei and nuclear spins situated on the protein. This is an intermediate step toward achieving the ultimate goal of NMR enhancement of the binding-site nuclei by polarization transfer from hyperpolarized (129)Xe. The hyperpolarized (129)Xe spectra resulting from exposure of four different proteins in the lyophilized, powdered form have been examined for evidence of binding. Each of the proteins, namely, metmyoglobin, methemoglobin, hen egg white lysozyme, and soybean lipoxygenase, yielded a distinctly different NMR line shape. With the exception of lysozyme, the proteins all possess a paramagnetic iron center which can be expected to rapidly relax the (129)Xe and produce a net shift in its resonance position if the noble gas atom occupies specific binding sites near the iron. At temperatures from 223 to 183 K, NMR signals were observed in the 0-40 ppm chemical shift range, relative to Xe in the gas phase. The signals broadened and shifted downfield as the temperature was reduced, indicating that Xe is exchanging between the gas phase and internal or external binding sites of the proteins. Additionally, conventional (129)Xe NMR studies of metmyoglobin and lipoxygenase in the solution state are presented. The temperature dependence of the chemical shift and line shape indicate exchange of Xe between adsorption sites on lipoxygenase and Xe in the solvent on the slow to intermediate exchange time scale. The NMR results are compared with N(2), Xe, and CH(4) gas adsorption isotherms. It is found that lipoxygenase is unique among the proteins studied in possessing a relatively high affinity for gas molecules, and in addition, demonstrating the most clearly resolved adsorbed (129)Xe NMR peak in the lyophilized state.

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Samples of diarrhoeic shellfish poison (DSP) mussels from several parts of the Italian Adriatic coastline were extracted and tested according to a number of different methods presently available, i.e. Yasumoto's mouse biotest, Kat's biotest, the ELISA test and the HPLC method. Results were compared for toxic levels detected in each sample. While a common qualitative result (toxic/non-toxic) was given by all the methods, no clear quantitative agreement was found. The differences between methods and consequent lack of agreement in results are discussed.

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The surface loop which in the Bacillus subtilis neutral protease (NP) extends from amino acid residue 188 to residue 194 was replaced, by site-directed mutagenesis, with the 10-residue segment which in the homologous polypeptide chain of thermolysin (TLN) binds calcium-4 [Matthews, B. W., Weaver, L. H., & Kester, W. R. (1974) J. Biol. Chem. 249, 8030-8044]. The mutant NP was isolated to homogeneity, and its structural, functional, calcium-binding, and stability properties were investigated. Proteolytic fragmentation with Staphylococcus aureus V8 protease of mutant NP was used to isolate and analyze the protein fragment encompassing the site of mutation, unambiguously establishing the effective insertion of the new 10-residue segment. Atomic absorption measurements allowed us to demonstrate that mutant NP binds three calcium ions instead of the two ions bound to wild-type NP, showing that indeed the chain segment grafted from TLN to NP maintains its calcium-binding properties. The mutant NP showed kinetic parameters essentially similar to those of the wild-type NP with Z-Phe-Leu-Ala-OH as substrate. The enzyme inactivation of mutant vs wild-type NP was studied as a function of free [Ca2+]. It was found that mutant NP was much less stable than the wild-type NP when enzyme solutions were dialyzed at neutral pH in the presence of [Ca2+] below 10(-3) M. On the other hand, the kinetic thermal stability to irreversible inactivation of mutant NP, when measured in the presence of 0.1 M CaCl2, was found to be increased about 2-fold over that of the wild-type NP. Thus, modulation of enzyme stability by free [Ca2+] in mutant NP correlates with similar findings previously reported for thermolysin. Overall, the results obtained indicate that protein engineering experiments can be used to prepare hybrid proteins on the basis of sequence and function analysis of homologous protein molecules and show the feasibility of engineering metal ion binding sites into proteins.

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The overall folding of neutral protease from Bacillus subtilis has been predicted by computer-aided modelling, taking as a basis the known three-dimensional structure of thermolysin. As expected from the 50% similarity of sequence between the two proteins, the structure of B. subtilis protease is similar to that of thermolysin, including the two-domain topology and location of elements of regular secondary structure (helices and strands), whereas specific differences were predicted in loop regions. A protruding and loose loop predicted in B. subtilis has been detected also experimentally by a limited proteolysis approach. Incubation of B. subtilis protease at pH 9.0 for 24 h at room temperature with trypsin at 20:1 ratio (by mass) leads to a specific and almost quantitative fission of the Arg214-Asn215 peptide bond located in a highly exposed, and thus probably flexible, loop of the protease. On the other hand, thermolysin was completely resistant to tryptic hydrolysis when reacted under identical conditions. The 'nicked' B. subtilis protease can be isolated by gel filtration chromatography at neutral pH, whereas the two constituting fragments 1-214 and 215-300 are separated under protein-denaturing conditions. Overall, these results indicate that the limited proteolysis approach can pinpoint a peculiar difference in surface structure between the two similar protein molecules of B. subtilis neutral protease and thermolysin and emphasize the potential use of proteolytic enzymes as structural probes of globular proteins.

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On the basis of the homology with the Bacillus thermoproteolyticus zinc endopeptidase thermolysin, we hypothesized that Glu-143 and His-231 are the key residues for the catalytic activity of the Bacillus subtilis neutral protease. To test this possibility by site-directed mutagenesis, we substituted these two residues with Ala, Ser, Trp and Arg, and Leu, Val and Cys respectively. All these substitutions dramatically affected the amount of secreted mutant proteins, as determined by immunological methods, and their catalytic activities. No appreciable secretion was observed with the three Glu mutants Trp, Ser and Arg, whereas the Glu----Ala mutant enzyme was secreted at a level of a few hundred micrograms per litre of culture. The His mutants were all secreted at higher levels (in the order of a few milligrams per litre) and their residual catalytic activity could be determined using Z-Ala-Leu-Ala as substrate. Our results confirm the key role played by Glu-143 and His-231 in catalysis and moreover suggest the existence of a relationship between the catalytic activity of the enzyme and the extent of its secretion. In this context, we present data suggesting an autoproteolytic mechanism of cleavage of the precursor form of the enzyme, analogous to the one previously reported for the B. subtilis subtilisin.

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Phenylalanine ammonia-lyase extracted form Rhodotorula rubra (IFO 1101) was immobilized into cellulose triacetate fibers made hemocompatible by physical blend with a platelet anti-aggregating agent. The entrapped enzyme could operate at physiological values of phenylalanine and tyrosine reducing their level to traces within a few hours. The optimum pH value for the entrapped enzyme shifted from 8.0 to 9.0. At blood pH the activity was about 68 per cent of the maximum. The entrapped enzyme retained its original activity in blood for more than 50 days.

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