The Prevalence of Children and Adolescents at Risk for Avoidant Restrictive Food Intake Disorder in a Pediatric and Adolescent Gynecology Clinic.

STUDY OBJECTIVE: The purpose of this study was to determine the prevalence of child and adolescent females at risk for Avoidant Restrictive Food Intake Disorder (ARFID) in a tertiary care pediatric and adolescent gynecology (PAG) clinic. DESIGN: Cross-sectional study design. SETTING: Tertiary care PAG clinic at the Hospital for Sick Children in Toronto, Ontario, Canada. PARTICIPANTS: Females between 8 and 18 years of age presenting to the tertiary care PAG clinic. INTERVENTION: Between October 2017 and April 2019, eligible patients completed a 3-part, self-administered questionnaire that included demographic and anthropometric information, reason(s) for referral, medical history, menstrual history and function, and the Eating Disorders in Youth-Questionnaire (EDY-Q). MAIN OUTCOME MEASURES: The main outcome measure was the prevalence of child and adolescent females who were identified to be at risk for ARFID in a tertiary care PAG clinic. RESULTS: Seven (3.7%) of 190 patients were identified to be at risk for ARFID based on the EDY-Q. All patients at risk for ARFID had a significantly lower body mass index (17.4 ± 1.6 vs 24.4 ± 6.7, P < .001) than patients not at risk for ARFID. CONCLUSIONS: This study demonstrated that 3.7% of patients seeking treatment in a tertiary care PAG clinic were identified to be at risk for ARFID. Clinicians in tertiary care PAG clinics can play a pivotal role in the identification and referral of children and adolescents at risk for ARFID. Referral to the patients' primary care physician or to an eating disorder program is important so as not to delay the diagnosis and treatment.

Anion-Caffeine Interactions Studied by 13C and 1H NMR and ATR-FTIR Spectroscopy.

This work investigates the interactions of a series of 11 anions with caffeine by utilizing 13C and 1H NMR and attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy. The aim of this study is to elucidate the molecular mechanisms of ion interactions with caffeine and to study how these interactions affect caffeine aggregation in aqueous solution. The chemical shift changes of caffeine 13C and 1H in the presence of salts provide a measure for anions' salting-out/salting-in abilities on individual carbon and hydrogen atoms in caffeine. The relative influences of anions on the chemical shift of individual atoms in the caffeine molecule are quantified. It is observed that strongly hydrated anions are excluded from the carbons on the six-member ring in caffeine and promote caffeine aggregation. On the other hand, weakly hydrated anions decrease caffeine aggregation by accumulating around the periphery of the caffeine molecule and binding to the ring structure. The ATR-FTIR results demonstrate that strongly hydrated anions desolvate the caffeine molecule and increase aggregation, while weakly hydrated anions have the opposite effects and salt caffeine into solution.

Hofmeister Ion-Induced Changes in Water Structure Correlate with Changes in Solvation of an Aggregated Protein Complex.

RecA is a naturally aggregating Escherichia coli protein that catalyzes the strand exchange reaction utilized in DNA repair. Previous studies have shown that the presence of salts influence RecA activity, aggregation, and stability and that salts stabilize RecA in an inverse-anionic Hofmeister series. Here we utilized attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy and circular dichroism (CD) to investigate how various Hofmeister salts alter the water structure and RecA solvation and aggregation. Spectroscopic studies performed in water and deuterium oxide suggest that salts alter water O-(1)H and O-(2)H stretch and bend vibrations as well as protein amide I (or I') and amide II (or II') vibrations. Anions have a much larger influence on water vibrations than cations. Water studies also show increased water-water and/or water-ion interactions in the presence of strongly hydrated SO4(2-) salts and evidence for decreased interactions with weakly hydrated Cl(-) and ClO4(-) salts. Salt-water difference infrared spectra show that kosmotropic salts are more hydrated than chaotropic salts. Interestingly, this is the opposite trend to the changes in protein solvation. Infrared spectra of RecA show that vibrations associated with protein desolvation were observed in the presence of SO4(2-) salts. Conversely, vibrations associated with protein solvation were observed in the presence of Cl(-) and ClO4(-) salts. Difference infrared studies on the dehydration of model proteins aided in identifying changes in RecA-solvent interactions. This study provides evidence that salt-induced changes in water vibrations correlate to changes in protein solvent interactions and thermal stability.

The effects of buffers and pH on the thermal stability, unfolding and substrate binding of RecA.

The Escherichia coli protein RecA is responsible for catalysis of the strand transfer reaction used in DNA repair and recombination. Previous studies in our lab have shown that high concentrations of salts stabilize RecA in a reverse-anionic Hofmeister series. Here we investigate how changes in pH and buffer alter the thermal unfolding and cofactor binding. RecA in 20mM HEPES, MES, Tris and phosphate buffers was studied in the pH range from 6.5 to 8.5 using circular dichroism (CD), infrared (IR) and fluorescence spectroscopies. The results show all of the buffers studied stabilize RecA up to 50°C above the Tris melting temperature and influence RecA's ability to nucleate on double-stranded DNA. Infrared and CD spectra of RecA in the different buffers do not show that secondary structural changes are associated with increased stability or decreased ability to nucleate on dsDNA. These results suggest the differences in stability arise from decreasing positive charge and/or buffer interactions.

Ion specific influences on the stability and unfolding transitions of a naturally aggregating protein; RecA.

The Escherichia coli RecA protein is a naturally aggregated protein complex that is affected by the presence of salts. In order to gain further insight into the nature of the ion-interactions on a naturally aggregating protein we used circular dichroism (CD), fluorescence and dynamic light scattering (DLS) to study the effects of different concentrations of MgCl2, CaCl2, NaCl, Na2SO4, and MgSO4 on RecA structure and thermal unfolding. The results show unique ion influences on RecA structure, aggregation, unfolding transitions and stability and the anion effects correlate with the reverse Hofmeister series. The mechanisms of the ion-induced changes most likely result from specific ion binding, changes in the interfacial tension and altered protein-solvent interactions that may be especially important for protein-protein interactions in naturally aggregating proteins. The presence of some ions leads to the formation of RecA complexes that are resistant to complete denaturation and nonspecific aggregation.

Infrared studies reveal unique vibrations associated with the PGK-ATP-3-PG ternary complex.

Phosphoglycerate kinase (PGK) catalyzes a reversible phospho-transfer reaction between ATP and 3-phosphoglycerate (3-PG) that is thought to require a hinge-bending motion in the protein that brings two separate substrate-binding domains together. We have used difference infrared spectroscopy to better understand the conformational changes that are unique to the PGK-ATP-3-PG complex. Caged nucleotides (caged-ADP and caged-ATP) were used to initiate nucleotide binding to PGK or PGK-3-PG complexes. The difference spectra include those of PGK-ATP minus PGK, PGK-3-PG-ATP minus PGK-3-PG, PGK-3-PG-ADP minus PGK-3-PG, and PGK-ADP minus PGK. The resulting spectra were compared in attempts to identify bands associated with each PGK complex. In addition, complementary activity assays were performed in the presence of caged-nucleotides. While PGK activity decreased in the presence of caged-ADP, the activity was not influenced by the addition of caged-ATP. The activity assay results suggest that the caged-ADP may interact with PGK substrate binding site(s) and inhibit phospho-transfer. Therefore, additional difference infrared nucleotide exchange experiments were used to isolate the differences between ADP and ATP binding to PGK. Difference FTIR spectra obtained on PGK-nucleotide-3-PG complexes show distinct bands that may result from amino acid side chains as well as structural changes in the hinge region and/or increased interactions such as salt bridges forming between the two domains. The infrared data obtained on the active ternary complexes show evidence of changes in alpha-helix and beta-structures as well as signals consistent with Arg, Asn, His, Lys, Asp, Glu, and additional side chains that are uniquely perturbed in the active ternary complex as compared to other PGK complexes.

Difference FTIR studies reveal nitrogen-containing amino acid side chains are involved in the allosteric regulation of RecA.

The Escherichia coli RecA protein performs the DNA strand-exchange reaction utilized in both genetic recombination and DNA repair. The binding of nucleotides triggers conformational changes throughout the protein resulting in the RecA-ATP (high DNA affinity) and RecA-ADP (low DNA affinity) structures. Difference infrared spectroscopy has allowed us to study protein structural changes in RecA that occur after binding ADP or ATP. Experiments were performed on control and uniformly (15)N-labeled RecA in an effort to assign vibrational changes to protein structures and study the molecular changes associated with the allosteric regulation of RecA. Comparison of RecA-ATP and RecA-ADP data indicates that the protein adopts unique secondary structures in each form and altered N-H stretching vibrations in the RecA-ADP structure not observed in the RecA-ATP data. Numerous vibrations throughout the 1700-1300 cm(-)(1) region are influenced by isotopic substitution and imply that many nitrogen-containing side chains are altered after ADP binds to RecA. The RecA-ATP data contain unique vibrations that are not observed in the RecA-ADP data and may be associated with Gln, Lys, Arg, or Asn. Model compound studies on control and (15)N-labeled glutamine and lysine provide additional evidence that supports the tentative assignments of vibrations observed in our difference spectra. In addition, we provide evidence that nitrogen-containing amino acids are important in locking in the low-DNA affinity, more compact conformation of the protein and that some of these interactions may not be present in a more extended, flexible RecA-ATP conformation.

Investigating structural changes induced by nucleotide binding to RecA using difference FTIR.

Nucleotide binding to RecA results in either the high-DNA affinity form (Adenosine 5'-triphosphate (ATP)-bound) or the more inactive protein conformation associated with a lower affinity for DNA (Adenosine 5'-diphosphate (ADP)-bound). Many of the key structural differences between the RecA-ATP and RecA-ADP bound forms have yet to be elucidated. We have used caged-nucleotides and difference FTIR in efforts to obtain a comprehensive understanding of the molecular changes induced by nucleotide binding to RecA. The photochemical release of nucleotides (ADP and ATP) from biologically inactive precursors was used to initiate nucleotide binding to RecA. Here we present ATP hydrolysis assays and fluorescence studies suggesting that the caged nucleotides do not interact with RecA before photochemical release. Furthermore, we now compare difference spectra obtained in H2O and D2O as our first attempt at identifying the origin of the vibrations influenced by nucleotide binding. The infrared data suggest that unique alpha-helical, beta structures, and side chain rearrangements are associated with the high- and low-DNA affinity forms of RecA. Difference spectra obtained over time isolate contributions arising from perturbations in the nucleotide phosphates and have provided further information about the protein structural changes involved in nucleotide binding and the allosteric regulation of RecA.

Time-resolved step-scan Fourier transform infrared spectroscopy of the CO adducts of bovine cytochrome c oxidase and of cytochrome bo(3) from Escherichia coli.

We have used cryogenic difference FTIR and time-resolved step-scan Fourier transform infrared (TR-FTIR) spectroscopies to explore the redox-linked proton-pumping mechanism of heme-copper respiratory oxidases. These techniques are used to probe the structure and dynamics of the heme a(3)-Cu(B) binuclear center and the coupled protein structures in response to the photodissociation of CO from heme Fe and its subsequent binding to and dissociation from Cu(B). Previous cryogenic (80 K) FTIR CO photodissociation difference results were obtained for cytochrome bo(3), the ubiquinol oxidase of Escherichia coli [Puustinen, A., et al. (1997) Biochemistry 36, 13195-13200]. These data revealed a connectivity between Cu(B) and glutamic acid E286, a residue which has been implicated in proton pumping. In the current work, the same phenomenon is observed using the CO adduct of bovine cytochrome aa(3) under cryogenic conditions, showing a perturbation of the equivalent residue (E242) to that in bo(3). Furthermore, using time-resolved (5 micros resolution) step-scan FTIR spectroscopy at room temperature, we observe the same spectroscopic perturbation in both cytochromes aa(3) and bo(3). In addition, we observe evidence for perturbation of a second carboxylic acid side chain, at higher frequency in both enzymes at room temperature. The high-frequency feature does not appear in the cryogenic difference spectra, indicating that the perturbation is an activated process. We postulate that the high-frequency IR feature is due to the perturbation of E62 (E89 in bo(3)), a residue near the opening of the proton K-channel and required for enzyme function. The implications of these results with respect to the proton-pumping mechanism are discussed. Finally, a fast loss of over 60% of the Cu(B)-CO signal in bo(3) is observed and ascribed to one or more additional conformations of the enzyme. This fast conformer is proposed to account for the uninhibited reaction with O(2) in flow-flash experiments.