Characterization of an archaeal inorganic pyrophosphatase from Sulfolobus islandicus using a [31P]-NMR-based assay.

Inorganic pyrophosphatase catalyzes the conversion of pyrophosphate to phosphate and is often critical for driving reactions forward in cellular processes such as nucleic acid and protein synthesis. Commonly used methods for quantifying pyrophosphatase enzyme activity employ reacting liberated phosphate with a second molecule to produce absorbance changes or employing a second enzyme in coupled reactions to produce a product with a detectable absorbance. In this investigation, a novel [31P]-NMR spectroscopy-based assay was used to quantitatively measure the formation of phosphate and evaluate the activity of inorganic pyrophosphatase from the thermoacidophilic Crenarchaeota Sulfolobus islandicus. The enzymatic activity was directly measured via integration of the [31P] resonance associated with the phosphate product (δ = 2.1 ppm). Sulfolobus islandicus inorganic pyrophosphatase preferentially utilized Mg2+ as divalent cation and had pH and temperature optimums of 6.0 of 50 °C, respectively. The Vmax value was 850 µmol/min/mg and the Km for pyrophosphate was 1.02 mM. Sequence analysis indicates the enzyme is a Family I pyrophosphatase. Sulfolobus islandicus inorganic pyrophosphatase was shown to be inhibited by sodium fluoride with a IC50 of 2.26 mM, compared to a IC50 of 0.066 mM for yeast inorganic pyrophosphatase. These studies reveal that a [31P]-NMR spectroscopy-based assay is an effective method for analyzing catalysis by phosphate-producing enzymes.

OBSERVED DEFICIENCIES IN MEDICAL STUDENT KNOWLEDGE OF TRANSGENDER AND INTERSEX HEALTH.

OBJECTIVE: Lesbian, gay, bisexual, transgender, and intersex (LGBTI) patients face many well-documented disparities in care which among transgender and intersex people can often be traced to providers' lack of knowledge. METHODS: We administered surveys to examine the self-assessed knowledge and attitudes of all medical students at Boston University regarding different LGBTI subpopulations. Survey questions were based on a Likert scale from 1 to 5; analysis was conducted with Wilcoxon rank sum tests. RESULTS: Overall there was a response rate of 24%, with the number of responses varying by class. Three of the 4 surveyed classes reported lower knowledge about transgender health than LGB health. Every class reported significantly lower knowledge of intersex health in comparison to LGB. Comfort with transgender or with intersex patients was lower than with LGB patients for all surveyed classes. Students across all self-identified groups (LGBTI, ally, not an ally) reported significantly lower average responses for knowledge and comfort regarding transgender or intersex health in comparison to that of LGB. Students in their preclinical years reported lower levels of knowledge in comparison with students in their clinical years. Students who identified as LGBTI reported significantly higher knowledge and comfort with only LGB and transgender health when compared with students who didn't identify as LGBTI. Respondents more frequently requested additional learning opportunities in transgender and intersex health than in LGB health. CONCLUSION: Self-reported knowledge of transgender and intersex health lags behind knowledge of LGB health, though these deficits appear partially responsive to targeted educational intervention. ABBREVIATIONS: BUSM = Boston University School of Medicine LGB = lesbian, gay, and bisexual LGBT = lesbian, gay, bisexual, and transgender LGBTI = lesbian, gay, bisexual, transgender, and intersex M1 = first-year medical student class M2 = second-year medical student class M3 = third-year medical student class M4 = fourth-year medical student class.

Patient Perspectives on Portal-Based Anxiety and Depression Screening in HIV Care: A Qualitative Study Using the Consolidated Framework for Implementation Research.

Electronic patient portals represent a promising means of integrating mental health assessments into HIV care where anxiety and depression are highly prevalent. Patient attitudes toward portal-based mental health screening within HIV clinics have not been well described. The aim of this formative qualitative study is to characterize the patient-perceived facilitators and barriers to portal-based anxiety and depression screening within HIV care in order to inform implementation strategies for mental health screening. Twelve adult HIV clinic patients participated in semi-structured interviews that were audio recorded and transcribed. The transcripts were coded using constructs from the Consolidated Framework for Implementation Research and analyzed thematically to identify the barriers to and facilitators of portal-based anxiety and depression screening. Facilitators included an absence of alternative screening methods, an approachable design, perceived adaptability, high compatibility with HIV care, the potential for linkage to treatment, an increased self-awareness of mental health conditions, the ability to bundle screening with clinic visits, and communicating an action plan for results. The barriers included difficulty navigating the patient portal system, a lack of technical support, stigmatization from the healthcare system, care team response times, and the novelty of using patient portals for communication. The patients in the HIV clinic viewed the use of a portal-based anxiety and depression screening tool as highly compatible with routine HIV care. Technical difficulties, follow-up concerns, and a fear of stigmatization were commonly perceived as barriers to portal use. The results of this study can be used to inform implementation strategies when designing or incorporating portal-based mental health screening into other HIV care settings.

Development of a new and reliable assay for choline kinase using 31P NMR.

Choline kinase catalyzes the conversion of choline to phosphocholine (PC) by transferring a phosphate group from adenosine triphosphate (ATP) as the first step in the biosynthetic pathway for the membrane phospholipid phosphatidylcholine, an essential pathway in the Leishmania parasitic protozoan. Commonly used methods for kinetically quantifying the enzyme include a radioisotope assay utilizing labeled choline and a coupled spectrophotometric assay with multiple enzymes and substrates that indirectly measures choline kinase activity. When testing potential inhibitors with the coupled assay, results can cast doubt on whether choline kinase is being inhibited or one of the coupled enzymes. Therefore, 31P NMR spectroscopy was used to quantitatively measure the formation of the key product, phosphocholine, and to evaluate choline kinase activity. Interrogation of 31P NMR spectroscopy offers a number of benefits. Since this isotope is 100% abundant and has a relatively large gyromagnetic ratio, it is considered one of the more sensitive nuclides. As such, the need for costly isotopic enriched phosphorous is not required and detection of the 31P signal is possible even at relatively low concentrations. The enzymatic activity of Leishmania infantum choline kinase was able to be directly measured via integration of the 31P resonance associated with the phosphocholine product (δ = 3.94 ppm). These initial studies reveal that a 31P NMR spectroscopic-based assay could be used for testing substrate or transition state analogs as competitive inhibitors of Leishmania choline kinase that may prevent phosphatidylcholine synthesis in the parasite.