Crystal structures of the human Dysferlin inner DysF domain

Background: Mutations in dysferlin, the first protein linked with the cell membrane repair mechanism, causes a group of muscular dystrophies called dysferlinopathies. Dysferlin is a type two-anchored membrane protein, with a single C terminal trans-membrane helix, and most of the protein lying in cytoplasm. Dysferlin contains several C2 domains and two DysF domains which are nested one inside the other. Many pathogenic point mutations fall in the DysF domain region. Results: We describe the crystal structure of the human dysferlin inner DysF domain with a resolution of 1.9 Angstroms. Most of the pathogenic mutations are part of aromatic/arginine stacks that hold the domain in a folded conformation. The high resolution of the structure show that these interactions are a mixture of parallel ring/guanadinium stacking, perpendicular H bond stacking and aliphatic chain packing. Conclusions: The high resolution structure of the Dysferlin DysF domain gives a template on which to interpret in detail the pathogenic mutations that lead to disease

House and Senate Vacancies: How Are They Filled?

Vacancies in Congress occur due to the death, resignation, or declination (refusal to serve) of a Senator or Representative, or as the result of expulsion or exclusion by either house. The Constitution requires that vacancies in both houses be filled by special election, but in the case of the Senate, it empowers state legislatures to provide for temporary appointments by the state governor until special elections can be scheduled. This report describes this process

'Dopamine-first' mechanism enables the rational engineering of the norcoclaurine synthase aldehyde activity profile

Norcoclaurine synthase (NCS) (EC 4.2.1.78) catalyzes the Pictet–Spengler condensation of dopamine and an aldehyde, forming a substituted (S)-tetrahydroisoquinoline, a pharmaceutically important moiety. This unique activity has led to NCS being used for both in vitro biocatalysis and in vivo recombinant metabolism. Future engineering of NCS activity to enable the synthesis of diverse tetrahydroisoquinolines is dependent on an understanding of the NCS mechanism and kinetics. We assess two proposed mechanisms for NCS activity: (a) one based on the holo X-ray crystal structure and (b) the ‘dopamine-first’ mechanism based on computational docking. Thalictrum flavum NCS variant activities support the dopamine-first mechanism. Suppression of the non-enzymatic background reaction reveals novel kinetic parameters for NCS, showing it to act with low catalytic efficiency. This kinetic behaviour can account for the ineffectiveness of recombinant NCS in in vivo systems, and also suggests NCS may have an in planta role as a metabolic gatekeeper. The amino acid substitution L76A, situated in the proposed aldehyde binding site, results in the alteration of the enzyme's aldehyde activity profile. This both verifies the dopamine-first mechanism and demonstrates the potential for the rational engineering of NCS activity

Structural and Contextual Patterns in Family Health History Knowledge among African American Adults: A Mixed-Methods Social Network Analysis Study*

Background: Family health history is a strong risk factor for many chronic diseases. Ethnic minorities have been found to have a low awareness of their family health history (FHH), which may pose a contributing factor to health disparities. Purpose: The purpose of this mixed-methods social network analysis study was to identify structural and contextual patterns in African American adults’ FHH knowledge based on interpersonal communication exchanges with their family members. Methods: African American adults completed individually administered family network interviews. Participants’ 3-generation family pedigree served as a visual aid to guide their interview. Our primary outcome of interest for this analysis was whether a family member was reported as someone who talks to the participant about their own (i.e., the family member’s) health, which we refer to as a “personal health informant.” To contextualize quantitative findings, participants were asked to describe how they learned about the health history of the relatives they identified during their interview. Results: Participants (n=37) reported an average family network size of 29.4 relatives (SD = 15.5; Range = 10-67). Each participant, on average, named 17% of their familial network as personal health informants. Multivariate regression results showed that participants were more likely to name an alter as a personal health informant if the alter was female (OR = 2.14, p = 0.0519), from the maternal side of the participant’s family (OR = 1.12, p = 0.0006), had one or more chronic health conditions (OR = 2.41, p = 0.0041), was someone who has discussions with the participant about the participant’s health (OR = 16.28, p < 0.0001), was a source of family health information (OR = 3.46, p = 0.0072), and was someone whose health the participant helps to monitor or track (OR = 5.93, p = 0.0002). Complementary qualitative findings indicate that FHH knowledge is facilitated by open, direct communication among relatives. Personal health informants were described as disclosing information for the purposes of informing others for preventive purposes and for gaining social support. Participants also learned about FHH via other methods, including direct observation, during caretaking, and following a relative’s death. Conclusions: Communication and disclosure practices is an important determinant of African Americans’ FHH knowledge. More culturally and contextually meaningful public health efforts are needed to promote family health history sharing, especially regarding paternal family health history, siblings, and extended relatives

Impact of the COVID-19 pandemic on chronic disease management and patient reported outcomes in patients with pulmonary hypertension: The Pulmonary Hypertension Association Registry

To better understand the impact of the COVID-19 pandemic on the care of patients with pulmonary hypertension, we conducted a retrospective cohort study evaluating health insurance status, healthcare access, disease severity, and patient reported outcomes in this population. Using the Pulmonary Hypertension Association Registry (PHAR), we defined and extracted a longitudinal cohort of pulmonary arterial hypertension (PAH) patients from the PHAR\u27s inception in 2015 until March 2022. We used generalized estimating equations to model the impact of the COVID-19 pandemic on patient outcomes, adjusting for demographic confounders. We assessed whether insurance status modified these effects via covariate interactions. PAH patients were more likely to be on publicly-sponsored insurance during the COVID-19 pandemic compared with prior, and did not experience statistically significant delays in access to medications, increased emergency room visits or nights in the hospital, or worsening of mental health metrics. Patients on publicly-sponsored insurance had higher healthcare utilization and worse objective measures of disease severity compared with privately insured individuals irrespective of the COVID-19 pandemic. The relatively small impact of the COVID-19 pandemic on pulmonary hypertension-related outcomes was unexpected but may be due to pre-established access to high quality care at pulmonary hypertension comprehensive care centers. Irrespective of the COVID-19 pandemic, patients who were on publicly-sponsored insurance seemed to do worse, consistent with prior studies highlighting outcomes in this population. We speculate that previously established care relationships may lessen the impact of an acute event, such as a pandemic, on patients with chronic illness

Interview with Half Century Club Inductees, Class of 1930

Oral history interview with Illinois State Normal University alumni, Class of 1930. The interview was conducted on May 10, 1980, by an unidentified interviewer. They discuss President Felmley, influential faculty, and racial discrimination experienced by students of color.https://ir.library.illinoisstate.edu/aoh/1002/thumbnail.jp

Structural Evidence for the Dopamine-First Mechanism of Norcoclaurine Synthase

Norcoclaurine synthase (NCS) is a Pictet-Spenglerase that catalyzes the first key step in plant benzylisoquinoline alkaloid metabolism, a compound family that includes bioactive natural products such as morphine. The enzyme has also shown great potential as a biocatalyst for the formation of chiral isoquinolines. Here we present new high-resolution X-ray crystallography data describing Thalictrum flavum NCS bound to a mechanism-inspired ligand. The structure supports two key features of the NCS "dopamine-first" mechanism: the binding of dopamine catechol to Lys-122 and the position of the carbonyl substrate binding site at the active site entrance. The catalytically vital residue Glu-110 occupies a previously unobserved ligand-bound conformation that may be catalytically significant. The potential roles of inhibitory binding and alternative amino acid conformations in the mechanism have also been revealed. This work significantly advances our understanding of the NCS mechanism and will aid future efforts to engineer the substrate scope and catalytic properties of this useful biocatalyst

The acceptance and kinetic resolution of alpha-Methyl Substituted Aldehydes by Norcoclaurine Synthase

Norcoclaurine synthase (NCS) catalyzes a stereoselective Pictet-Spengler reaction to give the key intermediate, (S)-norcoclaurine in benzylisoquinoline alkaloid (BIA) biosynthesis. This family of alkaloids contains many bioactive molecules including morphine and berberine. Recently, NCS has been demonstrated to accept a variety of aldehydes and some ketones as substrates, leading to a range of chiral tetrahydroisoquinoline (THIQ) products. Here, we report the unusual acceptance of a-substituted aldehydes, in particular a-methyl substituted aldehydes, by wild-type Thalictrum flavum NCS (Δ33TfNCS) to give THIQ products. Moreover, the kinetic resolution of several a-substituted aldehydes to give THIQs with two defined chiral centers in a single step with high conversions was achieved. Several dopamine analogues were also accepted as substrates and reactions were amenable to scale-up. Active site mutants of TfNCS were then used which demonstrated the potential to enhance the stereoselectivities in the reaction and improve yields. Rationale for the acceptance of these substrates and improved activity with different mutants has been gained from a co-crystallized structure of Δ33TfNCS with a non-productive mimic of a reaction intermediate bound in the active site. Finally, molecular dynamics simulations were performed to study the binding of dopamine and an a-substituted aldehyde and provided further insight into the reaction with these substrates

Single step syntheses of (1 S)-aryltetrahydroisoquinolines by norcoclaurine synthases

The 1-aryl-tetrahydroisoquinoline (1-aryl-THIQ) moiety is found in many biologically active molecules. Single enantiomer chemical syntheses are challenging and although some biocatalytic routes have been reported, the substrate scope is limited to certain structural motifs. The enzyme norcoclaurine synthase (NCS), involved in plant alkaloid biosynthesis, has been shown to perform stereoselective Pictet–Spengler reactions between dopamine and several carbonyl substrates. Here, benzaldehydes are explored as substrates and found to be accepted by both wild-type and mutant constructs of NCS. In particular, the variant M97V gives a range of (1 S)-aryl-THIQs in high yields (48–99%) and e.e.s (79–95%). A cocrystallised structure of the M97V variant with an active site reaction intermediate analogue is also obtained with the ligand in a pre-cyclisation conformation, consistent with (1 S)-THIQs formation. Selected THIQs are then used with catechol O-methyltransferases with exceptional regioselectivity. This work demonstrates valuable biocatalytic approaches to a range of (1 S)-THIQ

Argyrin B a non-competitive inhibitor of the human immunoproteasome exhibiting preference for β1i

Inhibitors of the proteasome have found broad therapeutic applications however, they show severe toxicity due to the abundance of proteasomes in healthy cells. In contrast, inhibitors of the immunoproteasome, which is upregulated during disease states, are less toxic and have increased therapeutic potential including against autoimmune disorders. In this project, we report argyrin B, a natural product cyclic peptide to be a reversible, non-competitive inhibitor of the immunoproteasome. Argyrin B showed selective inhibition of the β5i and β1i sites of the immunoproteasome over the β5c and β1c sites of the constitutive proteasome with nearly 20-fold selective inhibition of β1i over the homologous β1c. Molecular modelling attributes the β1i over β1c selectivity to the small hydrophobic S1 pocket of β1i and β5i over β5c to site-specific amino acid variations that enable additional bonding interactions and stabilization of the binding conformation. These findings facilitate the design of immunoproteasome selective and reversible inhibitors that may have a greater therapeutic potential and lower toxicity