Bio-Behavioral Changes Following Transition to Automated Insulin Delivery: A Large Real-Life Database Analysis

   Objective: Document glycemic and user-initiated bolus changes following transition from predictive-low glucose suspend (PLGS) system to automated insulin delivery (AID) system during real-life use. Research Design and Methods: Analysis of 2,329,166 days (6,381 patient-years) of continuous glucose monitoring (CGM) and insulin therapy data for 19,354 individuals with Type 1 Diabetes, during 1-month PLGS (Basal-IQ technology) use followed by 3-month AID use (Control-IQ technology). Baseline characteristics: 55.4 percent female, age (median/quartiles/range) 39/19-58/1-92 years, glucose management indicator (GMI) 7.5±0.8. Primary outcome: time in target range (TIR 70-180mg/dL). Secondary outcomes: CGM-based glycemic control metrics; frequency of user-initiated boluses. Results: Compared to PLGS, AID increased TIR on average from 58.4 to 70.5 percent. GMI and percent time above/below target range improved as well, 7.5 to 7.1; 39.9 to 28.1 percent, and 1.66 to 1.46 percent, respectively, all p-levels 8.0 (TIR improvement 13.2 percentage points). User-initiated correction boluses decreased from 2.7 to 1.8 per day, while user-initiated meal boluses remained stable at 3.6 to 3.8 per day. Conclusions: Observed in real life of over 19,000 individuals with type 1 diabetes, transitions from PLGS to AID resulted in improvement of all glycemic parameters, equivalent to improvements observed in randomized clinical trials, and reduced user-initiated boluses.  However, glycemic and behavioral changes with AID use may differ greatly across different demographic and clinical groups. </p

Exposing the Alkanesulfonate Monooxygenase Protein–Protein Interaction Sites

The alkanesulfonate monooxygenase enzymes (SsuE and SsuD) catalyze the desulfonation of diverse alkanesulfonate substrates. The SsuE enzyme is an NADPH-dependent FMN reductase that provides reduced flavin to the SsuD monooxygenase enzyme. Previous studies have highlighted the presence of protein–protein interactions between SsuE and SsuD thought to be important in the flavin transfer event, but the putative interaction sites have not been identified. Protected sites on specific regions of SsuE and SsuD were identified by hydrogen–deuterium exchange mass spectrometry. An α-helix on SsuD containing conserved charged amino acids showed a decrease in percent deuteration in the presence of SsuE. The α-helical region of SsuD is part of an insertion sequence and is adjacent to the active site opening. A SsuD variant containing substitutions of the charged residues showed a 4-fold decrease in coupled assays that included SsuE to provide reduced FMN, but there was no activity observed with an SsuD variant containing a deletion of the α-helix under similar conditions. Desulfonation by the SsuD deletion variant was only observed with an increase in enzyme and substrate concentrations. Although activity was observed under certain conditions, there were no protein–protein interactions observed with the SsuD variants and SsuE in pull-down assays and fluorimetric titrations. The results from these studies suggest that optimal transfer of reduced flavin from SsuE to SsuD requires defined protein–protein interactions, but diffusion can occur under specified conditions. A basis is established for further studies to evaluate the structural features of the alkanesulfonate monooxygenase enzymes that promote desulfonation

Changes in Protein Dynamics in <i>Escherichia coli</i> SufS Reveal a Possible Conserved Regulatory Mechanism in Type II Cysteine Desulfurase Systems

In the Suf Fe–S cluster assembly pathway, the activity of the cysteine desulfurase, SufS, is regulated by interactions with the accessory sulfotransferase protein, SufE. SufE has been shown to stimulate SufS activity, likely by inducing conformational changes in the SufS active site that promote the desulfurase step and by acting as an efficient persulfide acceptor in the transpersulfuration step. Previous results point toward an additional level of regulation through a “half-sites” mechanism that affects the stoichiometry and affinity for SufE as the dimeric SufS shifts between desulfurase and transpersulfuration activities. Investigation of the covalent persulfide intermediate of SufS by backbone amide hydrogen–deuterium exchange mass spectrometry identified two active site peptides (residues 225–236 and 356–366) and two peptides at the dimer interface of SufS (residues 88–100 and 243–255) that exhibit changes in deuterium uptake upon formation of the intermediate. Residues in these peptides are organized to form a conduit between the two active sites upon persulfide formation and include key cross-monomer interactions, suggesting they may play a role in the half-sites regulation. Three evolutionarily conserved residues at the dimer interface (R92, E96, and E250) were investigated by alanine scanning mutagenesis. Two of the substituted enzymes (E96A and E250A SufS) resulted in 6-fold increases in the value of <i>K</i>_SufE, confirming a functional role. Re-examination of the dimer interface in reported crystal structures of SufS and the SufS homologue CsdA identified previously unnoticed residue mobility at the dimer interface. The identification of conformational changes at the dimer interface by hydrogen–deuterium exchange confirmed by mutagenesis and structural reports provides a physical mechanism for active site communication in the half-sites regulation of SufS activity. Given the conservation of the interface interactions, this mechanism may be broadly applicable to type II cysteine desulfurase systems