Surf4 (Erv29p) binds amino-terminal tripeptide motifs of soluble cargo proteins with different affinities, enabling prioritization of their exit from the endoplasmic reticulum.

Some secreted proteins that assemble into large complexes, such as extracellular matrices or hormones and enzymes in storage granules, must be kept at subaggregation concentrations during intracellular trafficking. We show surfeit locus protein 4 (Surf4) is the cargo receptor that establishes different steady-state concentrations for a variety of soluble cargo proteins within the endoplasmic reticulum (ER) through interaction with the amino-terminal tripeptides exposed after removal of leader sequences. We call this motif the ER-Exit by Soluble Cargo using Amino-terminal Peptide-Encoding motif (ER-ESCAPE motif). Proteins that most readily aggregate in the ER lumen (e.g., dentin sialophosphoprotein [DSPP] and amelogenin, X-linked [AMELX]) have strong ER-ESCAPE motifs to inhibit aggregate formation, while less susceptible cargo exhibits weaker motifs. Specific changes in a single amino acid of the tripeptide result in aggregate formation and failure to efficiently traffic cargo out of the ER. A logical subset of 8,000 possible tripeptides starting a model soluble cargo protein (growth hormone) established a continuum of steady-state ER concentrations ranging from low (i.e., high affinity for receptor) to the highest concentrations associated with bulk flow-limited trafficking observed for nonbinding motifs. Human cells lacking Surf4 no longer preferentially trafficked cargo expressing strong ER-ESCAPE motifs. Reexpression of Surf4 or expression of yeast's ortholog, ER-derived vesicles protein 29 (Erv29p), rescued enhanced ER trafficking in Surf4-null cells. Hence our work describes a new way of preferentially exporting soluble cargo out of the ER that maintains proteins below the concentrations at which they form damaging aggregates.

Effect of genetic background on the dystrophic phenotype in mdx mice.

Genetic background significantly affects phenotype in multiple mouse models of human diseases, including muscular dystrophy. This phenotypic variability is partly attributed to genetic modifiers that regulate the disease process. Studies have demonstrated that introduction of the γ-sarcoglycan-null allele onto the DBA/2J background confers a more severe muscular dystrophy phenotype than the original strain, demonstrating the presence of genetic modifier loci in the DBA/2J background. To characterize the phenotype of dystrophin deficiency on the DBA/2J background, we created and phenotyped DBA/2J-congenic Dmdmdx mice (D2-mdx) and compared them with the original, C57BL/10ScSn-Dmdmdx (B10-mdx) model. These strains were compared with their respective control strains at multiple time points between 6 and 52 weeks of age. Skeletal and cardiac muscle function, inflammation, regeneration, histology and biochemistry were characterized. We found that D2-mdx mice showed significantly reduced skeletal muscle function as early as 7 weeks and reduced cardiac function by 28 weeks, suggesting that the disease phenotype is more severe than in B10-mdx mice. In addition, D2-mdx mice showed fewer central myonuclei and increased calcifications in the skeletal muscle, heart and diaphragm at 7 weeks, suggesting that their pathology is different from the B10-mdx mice. The new D2-mdx model with an earlier onset and more pronounced dystrophy phenotype may be useful for evaluating therapies that target cardiac and skeletal muscle function in dystrophin-deficient mice. Our data align the D2-mdx with Duchenne muscular dystrophy patients with the LTBP4 genetic modifier, making it one of the few instances of cross-species genetic modifiers of monogenic traits.

Structure of Quinolinate Synthase from Pyrococcus horikoshii in the Presence of Its Product, Quinolinic Acid.

Quinolinic acid (QA) is a common intermediate in the biosynthesis of nicotinamide adenine dinucleotide (NAD(+)) and its derivatives in all organisms that synthesize the molecule de novo. In most prokaryotes, it is formed from the condensation of dihydroxyacetone phosphate (DHAP) and aspartate-enamine by the action of quinolinate synthase (NadA). NadA contains a [4Fe-4S] cluster cofactor with a unique, non-cysteinyl-ligated, iron ion (Fea), which is proposed to bind the hydroxyl group of a postulated intermediate in the last step of the reaction to facilitate a dehydration. However, direct evidence for this role in catalysis has yet to be provided. Herein, we present the structure of NadA in the presence of the product of its reaction, QA. We find that N1 and the C7 carboxylate group of QA ligate to Fea in a bidentate fashion, which is confirmed by Hyperfine Sublevel Correlation (HYSCORE) spectroscopy. This binding mode would place the C5 hydroxyl group of the postulated final intermediate distal to Fea and virtually incapable of coordinating to it. The structure shows that three strictly conserved amino acids, Glu198, Tyr109, and Tyr23, are in close proximity to the bound product. Substitution of these amino acids with Gln, Phe, and Phe, respectively, leads to complete loss of activity.

Characterization of BciB: a ferredoxin-dependent 8-vinyl-protochlorophyllide reductase from the green sulfur bacterium Chloroherpeton thalassium.

Two enzymes, BciA and BciB, are known to reduce the C-8 vinyl group of 8-vinyl protochlorophyllide, producing protochlorophyllide a, during the synthesis of chlorophylls and bacteriochlorophylls in chlorophototrophic bacteria. BciA from the green sulfur bacterium Chlorobaculum tepidum reduces the C-8 vinyl group using NADPH as the reductant. Cyanobacteria and some other chlorophototrophs have a second, nonhomologous type of 8-vinyl reductase, BciB, but the biochemical properties of this enzyme have not yet been described. In this study, the bciB gene of the green sulfur bacterium Chloroherpeton thalassium was expressed in Escherichia coli , and the recombinant protein was purified and characterized. Recombinant BciB binds a flavin adenine dinucleotide cofactor, and EPR spectroscopy as well as quantitative analyses of bound iron and sulfide suggest that BciB binds two [4Fe-4S] clusters, one of which may not be essential for the activity of the enzyme. Using electrons provided by reduced ferredoxin or dithionite, recombinant BciB was active and reduced the 8-vinyl moiety of the substrate, 8-vinyl protochlorophyllide, producing protochlorophyllide a. A structural model for BciB based on a recent structure for the FrhB subunit of F420-reducing [NiFe]-hydrogenase of Methanothermobacter marburgensis is proposed. Possible reasons for the occurrence and distribution of BciA and BciB among various chlorophototrophs are discussed.

Effect of a Trauma-Awareness Course on Teachers' Perceptions of Conflict With Preschool-Aged Children From Low-Income Urban Households: A Cluster Randomized Clinical Trial.

Importance: Adverse childhood experiences are common and are associated with changes in early development and learning, but training early childhood educators in trauma-informed approaches to care has not been evaluated with randomized clinical trials. Objective: To determine whether a 6-session (12-week) professional development course, "Enhancing Trauma Awareness," improved the quality of teachers' relationships with the children in their classrooms. Design, Setting, and Participants: This cluster randomized clinical trial conducted from September 2017 to May 2018 allocated classrooms by a computer-generated random sequence to intervention (attend Enhancing Trauma Awareness course) and control (no course) groups. Outcomes were reported by participants via survey and analyzed by group allocation. Classrooms under the auspice of the School District of Philadelphia, Pennsylvania, serving 3- and 4-year-old children living in low-income households were invited to participate. Lead and/or assistant teachers from 63 of 348 eligible classrooms (18.1%) agreed to participate, and none were excluded. Of 96 enrolled teachers, 93 (96.9%) were assessed at follow-up (61 of 63 classrooms [96.8%]). Exposures: In September 2017, 32 classrooms (48 teachers) were assigned to receive a professional development course that taught about the effects of trauma using a group-based relational process, and 31 classrooms (48 teachers) received no intervention. Teachers completed online surveys immediately before and after the course. Exploratory focus groups with intervention teachers (n = 15) were conducted 5 months after the course ended. Main Outcomes and Measures: The primary outcome was teacher-children relationship quality, with a hypothesized decrease in teacher-children conflict scores. Secondary outcomes included relational capacities (eg, empathy, emotion regulation, and dispositional mindfulness). Focus group themes described teachers' experience of the course. Results: Of 96 teachers enrolled, 93 (96.9%) were women, and 58 (60.4%) were 40 years and older. Follow-up surveys were completed by 46 teachers (95.8%) in the control group and 47 (97.9%) in the intervention group, of whom 38 (79.2%) attended 4 or more course sessions. Adjusting for baseline values, mean (SE) conflict scores after the course were not significantly different between course participants (15.8 [0.6]) and controls (15.0 [0.6]) (effect size = 0.16; 95% CI, -0.19 to 0.52). There were no significant between-group differences in secondary outcomes. However, in focus groups, the teachers reported improvements in teacher-children relationship quality and several related relational capacities. Conclusions and Relevance: A course to enhance trauma awareness among preschool teachers did not reduce teacher-children conflict scores, yet qualitative assessments suggested the potential for improved teacher-children relationship quality. Trial Registration: ClinicalTrials.gov identifier: NCT03303482.

Regulation of the activity of Escherichia coli quinolinate synthase by reversible disulfide-bond formation.

Quinolinate synthase (NadA) catalyzes a unique condensation reaction between dihydroxyacetone phosphate and iminoaspartate, yielding inorganic phosphate, 2 mol of water, and quinolinic acid, a central intermediate in the biosynthesis of nicotinamide adenine dinucleotide and its derivatives. The enzyme from Escherichia coli contains a C (291)XXC (294)XXC (297) motif in its primary structure. Bioinformatics analysis indicates that only Cys297 serves as a ligand to a [4Fe-4S] cluster that is required for turnover. In this report, we show that the two remaining cysteines, Cys291 and Cys294, undergo reversible disulfide-bond formation, which regulates the activity of the enzyme. This mode of redox regulation of NadA appears physiologically relevant, since disulfide-bond formation and reduction are effected by oxidized and reduced forms of E. coli thioredoxin. A midpoint potential of -264 +/- 1.77 mV is approximated for the redox couple.

Characterization of quinolinate synthases from Escherichia coli, Mycobacterium tuberculosis, and Pyrococcus horikoshii indicates that [4Fe-4S] clusters are common cofactors throughout this class of enzymes.

Quinolinate synthase (NadA) catalyzes a unique condensation reaction between iminoaspartate and dihydroxyacetone phosphate, affording quinolinic acid, a central intermediate in the biosynthesis of nicotinamide adenine dinucleotide (NAD). Iminoaspartate is generated via the action of l-aspartate oxidase (NadB), which catalyzes the first step in the biosynthesis of NAD in most prokaryotes. NadA from Escherichia coli was hypothesized to contain an iron-sulfur cluster as early as 1991, because of its observed labile activity, especially in the presence of hyperbaric oxygen, and because its primary structure contained a CXXCXXC motif, which is commonly found in the [4Fe-4S] ferredoxin class of iron-sulfur (Fe/S) proteins. Indeed, using analytical methods in concert with Mossbauer and electron paramagnetic resonance spectroscopies, the protein was later shown to harbor a [4Fe-4S] cluster. Recently, the X-ray structure of NadA from Pyrococcus horikoshii was solved to 2.0 A resolution [Sakuraba, H., Tsuge, H.,Yoneda, K., Katunuma, N., and Ohshima, T. (2005) J. Biol. Chem. 280, 26645-26648]. This protein does not contain a CXXCXXC motif, and no Fe/S cluster was observed in the structure or even mentioned in the report. Moreover, rates of quinolinic acid production were reported to be 2.2 micromol min (-1) mg (-1), significantly greater than that of E. coli NadA containing an Fe/S cluster (0.10 micromol min (-1) mg (-1)), suggesting that the [4Fe-4S] cluster of E. coli NadA may not be necessary for catalysis. In the study described herein, nadA genes from both Mycobacterium tuberculosis and Pyrococcus horikoshii were cloned, and their protein products shown to contain [4Fe-4S] clusters that are absolutely required for activity despite the absence of a CXXCXXC motif in their primary structures. Moreover, E. coli NadA, which contains nine cysteine residues, is shown to require only three for turnover (C113, C200, and C297), of which only C297 resides in the CXXCXXC motif. These results are consistent with a bioinformatics analysis of NadA sequences, which indicates that three cysteines are strictly conserved across all species. This study concludes that all currently annotated quinolinate synthases harbor a [4Fe-4S] cluster, that the crystal structure reported by Sakuraba et al. does not accurately represent the active site of the protein, and that the "activity" reported does not correspond to quinolinate formation.

Mobile phone-based remote patient monitoring system for management of hypertension in diabetic patients.

BACKGROUND: Rising concern over the poor level of blood-pressure (BP) control among hypertensive patients has prompted searches for novel ways of managing hypertension. The objectives of this study were to develop and pilot-test a home BP tele-management system that actively engages patients in the process of care. METHODS: Phase 1 involved a series of focus-group meetings with patients and primary care providers to guide the system's development. In Phase 2, 33 diabetic patients with uncontrolled ambulatory hypertension were enrolled in a 4-month pilot study, using a before-and-after design to assess its effectiveness in lowering BP, its acceptability to users, and the reliability of home BP measurements. RESULTS: The system, developed using commodity hardware, comprised a Bluetooth-enabled home BP monitor, a mobile phone to receive and transmit data, a central server for data processing, a fax-back system to send physicians' reports, and a BP alerting system. In the pilot study, 24-h ambulatory BP fell by 11/5 (+/-13/7 SD) mm Hg (both P < .001), and BP control improved significantly. Substantially more home readings were received by the server than expected, based on the preset monitoring schedule. Of 42 BP alerts sent to patients, almost half (n = 20) were due to low BP. Physicians received no critical BP alerts. Patients perceived the system as acceptable and effective. CONCLUSIONS: The encouraging results of this study provide a strong rationale for a long-term, randomized, clinical trial to determine whether this home BP tele-management system improves BP control in the community among patients with uncontrolled hypertension.